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[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "Japanese encephalitis virus JEV , an arthropod-borne flavivirus, is a major cause of acute viral encephalitis in humans. No approved drug is available for the specific treatment of JEV infections, and the available vaccines are not effective against all clinical JEV isolates. In the study described here, a high-throughput screening of an FDA-approved drug library for inhibitors of JEV was performed. Five hit drugs that inhibited JEV infection with a selective index of >10 were identified. The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized.", "The antiviral activities of these five hit drugs against other flavivirus, including Zika virus, were also validated. As three of the five hit drugs were calcium inhibitors, additional types of calcium inhibitors that confirmed that calcium is essential for JEV infection, most likely during viral replication, were utilized. Adaptive mutant analysis uncovered that replacement of Q130, located in transmembrane domain 3 of the nonstructural NS4B protein, which is relatively conserved in flaviviruses, with R or K conferred JEV resistance to manidipine, a voltage-gated Ca 2+ channel VGCC inhibitor, without an apparent loss of the viral growth profile. Furthermore, manidipine was indicated to protect mice against JEV-induced lethality by decreasing the viral load in the brain, while it abrogated the histopathological changes associated with JEV infection. This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses.", "This study provides five antiflavivirus candidates and identifies cytoplasmic calcium to be a novel antiviral target for the treatment of JEV infection. The findings reported here provide therapeutic possibilities for combating infections caused by flaviviruses. IMPORTANCE No approved therapy for the treatment of Japanese encephalitis virus infection is currently available. Repurposing of approved drugs would accelerate the development of a therapeutic stratagem. In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant.", "In this study, we screened a library of FDA-approved drugs and identified five hit drugs, especially calcium inhibitors, exerting antiflavivirus activity that blocked viral replication. The in vivo efficacy and toxicity of manidipine were investigated with a mouse model of JEV infection, and the viral target was identified by generating an adaptive mutant. Text: F laviviruses are taxonomically classified in the genus Flavivirus and family Flaviviridae. These viruses comprise over 70 different pathogens, such as Japanese encephalitis virus JEV , Zika virus ZIKV , dengue virus DENV , West Nile virus WNV , and yellow fever virus YFV . Most flaviviruses are arthropod borne and cause public health problems worldwide . .", "Most flaviviruses are arthropod borne and cause public health problems worldwide . . The development and usage of vaccines against some flaviviruses, such as JEV, YFV, and tick-borne encephalitis virus TBEV , have decreased the rates of morbidity and mortality from infections caused by these viruses . ; however, flavivirus-induced diseases are still pandemic, and few therapies beyond intensive supportive care are currently available. Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 .", "Flaviviruses have an approximately 11-kb positive-stranded RNA genome containing a single open reading frame ORF flanked by untranslated regions UTRs at both termini. The ORF encodes three structural proteins, including the capsid C , membrane premembrane prM and membrane M , and envelope E , and seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 . . These seven nonstructural proteins participate in viral replication, virion assembly, and virus escape from immune surveillance. To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs.", "To date, no specific antivirals with activity against flaviviruses are available. To address this, we conducted a screen of a library of 1,018 FDA-approved drugs. Since flaviviruses are similar in structure and pathogenesis, we first utilized JEV as the prototype to screen the drug library and subsequently validated the antiviral activities with ZIKV, WNV, and DENV type 2 DENV-2 . The hit drugs identified in this study offer potential new therapies for the treatment of flavivirus infection and disease. Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay .", "Screening of an FDA-approved drug library for inhibitors of JEV infection. Recombinant viral particles RVPs with the luciferase-reporting replicon enveloped by the JEV structural proteins were used to select inhibitors, with a focus on those that inhibit virus entry and replication, by a high-throughput screening HTS assay . . The number of genomic RNA copies of RVP was determined to be 8.4 ϫ 10 6 copies/ml by using a standard curve generated with plasmids carrying the infectious clone. The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds.", "The HTS assay conditions, including the seeding cell density and RVP dose, were optimized to be 10,000 cells per 96-well plate and 20 l 16 copies/cell RVP for the infective dose, respectively. Under the optimized conditions, the signal-to-basal S/B ratio, coefficient of variation CV , and Z= factor were 38,374, 2.8%, and 0.89, respectively, which demonstrated that the assay was robust and suitable for the large-scale screening of compounds. A schematic of the HTS assay is depicted in Fig. 1B . After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig.", "After three rounds of screening, five hits with a selective index SI; which is equal to the 50% cytotoxic concentration CC 50 /50% inhibitory concentration IC 50 of Ͼ10 were selected. The CC 50 values of the hit drugs exhibited in Fig. 1B were similar to those previously published for diverse cell systems but determined using different toxicity assays . . . . . . . . . Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker.", ". Three of the hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were dihydropyridine DHP voltage-gated Ca 2ϩ channel VGCC antagonists, while pimecrolimus is an inhibitor of inflammatory cytokine secretion and nelfinavir mesylate is an HIV-1 protease blocker. All five drugs exhibited a dose-dependent inhibition of JEV RVP infection Fig. 1C . To validate the antiviral effect, hit drugs were purchased from other commercial sources and tested. In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs.", "In the reconfirmation screen, all hit drugs showed antiviral and cytotoxic effects similar to those found in the primary screen. Validation of hit drugs. To verify the results obtained by the luciferase reporter assays, we also investigated the antiviral effect of the five hit drugs on wild-type JEV strain AT31. As expected from the HTS assay, all five drugs robustly inhibited virus production, with a reduction of approximately 4 to 5 log units at the highest concentration and an approximately 1-log-unit decrease with 2.5 M the drugs Fig. 2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C .", "2B . A sharp decrease in JEV RNA levels was also detected Fig. 2C . The attenuated RNA levels in the high-dose, middle-dose, and low-dose groups were all above 40%. In particular, in the manidipine-treated group, the inhibitory effect was at least 80% compared to that for the control, which showed a strong inhibition of viral replication. Consistent with the inhibition of virus replication and production, expression of the viral structural protein prM was hardly detectable following treatment with the drugs at the high concentration Fig. 2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro.", "2D . Overall, the results in Fig. 2 confirmed that the five hit drugs inhibited JEV infection in a dose-dependent manner in vitro. Drugs inhibit JEV infection during viral RNA synthesis. Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig.", "Because RVPs, which have a natural virus-like envelope on the outside and a replicon on the inside, permitted the quantification of JEV productive entry and replication, a time-of-addition experiment was performed to investigate whether the hit drugs blocked the entry step or the replication step. As shown in Fig. 3B , no suppression of luciferase activity by any of the hit drugs was observed when they were used as treatments before infection or during infection or as a virucide, suggesting that these drugs do not inhibit JEV infection either by inactivating the virus directly or by blocking JEV entry. However, these drugs exerted fully inhibitory effects when they were added at 1 h postinfection, suggesting that viral replication was the stage at which these drugs showed inhibitory activity. To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively.", "To confirm this suggestion, we investigated the inhibitory effects of these drugs on the JEV replicon. The highest concentration of manidipine and nelfinavir mesylate tested in baby hamster kidney BHK-21 cells was adjusted to 5 M and 10 M, respectively. It was shown that all five drugs inhibited JEV RNA synthesis in a dosedependent manner, while neither drug inhibited the initial translation of replicon RNA . Fig. 3C , confirming that these drugs inhibited JEV infection at the stage of replication. Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV.", "Hit drugs exhibit broad-spectrum antiflavivirus activity. In order to determine whether the antiviral activity of the five hit drugs extended to other flaviviruses, we explored their antiviral effect against ZIKV. Similar to the findings for JEV, the ZIKV titer was decreased by multiple log units when ZIKV was treated with a high concentration of each of the drugs Fig. 4A . Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A .", "Moreover, ZIKV exhibited a higher sensitivity to the two calcium channels inhibitors manidipine and cilnidipine than JEV, with no plaque formation being observed at 10 M. Consistent with this result, sharp decreases in the level of replication of ZIKV RNA and the level of expression of viral protein were also detected Fig. 4A . Notably, treatment with 5 M manidipine produced a 95% inhibition of viral replication, translation, and viral yields. Taken together, these results indicate that the hit drugs could effectively inhibit ZIKV infection. Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments.", "Since these drugs exhibited their anti-JEV effects at the stage of viral replication, we further tested the effects against WNV and DENV-2 by using WNV and DENV-2 replicons. Similar to the results for JEV, a dose-dependent reduction in the level of WNV replication was observed with the drug treatments. The same phenotype was observed for DENV-2 for all drugs except nelfinavir mesylate, which showed no effect at the concentrations tested Fig. 4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors.", "4B and C . Together, these results indicate that the five hit drugs are excellent candidates for broad-spectrum antiflavivirus treatment. Antiviral effect of calcium inhibitors. Since three hit drugs, manidipine, cilnidipine, and benidipine hydrochloride, were DHP VGCC inhibitors, we asked whether other calcium antagonists could block JEV infection. To address this question, we employed four different classes of inhibitors. Verapamil, a prototype phenylalkylamine PAA VGCC inhibitor . , exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection.", ", exhibited a dose-dependent inhibition of JEV on both African Green monkey kidney Vero and human hepatocellular carcinoma Huh-7 cells Fig. 5 , which was consistent with the inhibitory effects of the DHP inhibitors, suggesting that calcium channels play an important role in JEV infection. Cyclosporine and 2-aminobiphenyl borate 2-APB , which inhibit the efflux of Ca 2ϩ from the mitochondrial and endoplasmic reticulum ER pool, respectively . . . . , were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection.", ", were also found to block JEV infection effectively. Similarly, treatment with the cell-permeant Ca 2ϩ chelator 1,2-bis- o-aminophenoxy -ethane-N,N,N=,N=-tetraacetic acid, tetraacetoxymethyl ester BAPTA-AM , could also suppress JEV infection. Taken together, we concluded that intracellular Ca 2ϩ is essential for JEV infection and cytoplasmic calcium is a potent target for antiflavivirus treatment. Selection and characterization of manidipine-resistant JEV. To identify the viral target of the calcium channel inhibitor, we selected a manidipine-resistant virus by serially passaging JEV in the presence of manidipine. Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A .", "Viruses from passage 20 P20 showed robust resistance compared with the wild type WT Fig. 6A . When JEV from P20 was treated with 5 M or 10 M manidipine, the viral titer was about 10-and 100-fold higher than that of the WT, respectively. Individual virus clones were isolated, and two isolates were randomly selected and amplified. An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B .", "An amino acid substitution was observed in two isolated clones, resulting in a glutamine Q -to-arginine R switch at amino acid position 130 in transmembrane domain 3 TMD3 of NS4B, i.e., position 2401 of the translated polyprotein in the JEV infectious cDNA clone Fig. 6B . Sequence alignment of NS4B indicated that Q130 was conserved in all flaviviruses except YFV, which possessed a lysine at that position Fig. 6B . The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown .", "The conserved Q130 of NS4B may account for the sensitivity of JEV, ZIKV, WNV, and DENV-2 to manidipine, as described above Fig. 4 , while YFV showed resistance to the drug data not shown . To confirm that the Q130R mutation did confer manidipine resistance and to investigate the role of Q130 in NS4B function, we produced JEV clones with the Q130R, Q130K, Q130E, or Q130A mutation by introducing the desired mutations into the infectious cDNA clone and rescuing the mutant viruses. To investigate the biological properties of the mutant viruses, we first examined the growth kinetics of the rescued viruses. As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection.", "As shown in Fig. 6C , all mutant viruses had an accumulation of infectious virions and reached the highest titer at 60 h postinfection. Infection of the Q130R and Q130K mutant viruses resulted in growth curves similar to the growth curve for the WT Fig. 6C , while the Q130E and Q130A mutants produced smaller amounts of viruses between 24 and 60 h. Analysis of the plaque morphology revealed that the plaques of the Q130R, Q130K, and Q130E mutants were similar to the plaques of the WT, whereas the plaques of the Q130A mutant were smaller than those of the WT. We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig.", "We next investigated the sensitivity of the four mutant viruses to manidipine. As shown in Fig. 6D , the Q130R and Q130K mutant viruses were resistant to manidipine. At a 10 M concentration, manidipine efficiently inhibited WT JEV infection and reduced the viral yields by approximately 4 log units, while the Q130R and Q130K mutant viruses were resistant to manidipine and the viral titer decreased less than 2 log units. The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth.", "The Q130A mutant virus demonstrated moderate resistance and a slightly higher Taken together, it could be concluded that Q130 not only is critical for conferring manidipine sensitivity but also is important for JEV replication. The replacement of glutamine with basic amino acids conferred resistance to manidipine without an apparent loss of growth. In vivo efficacy of manidipine. As manidipine exhibited the strongest inhibitory activities on JEV replication as well as ZIKV infection when its activities were compared with those of the five hit drugs Fig. 2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection.", "2 and 4A , we further examined the protective effect of manidipine against JEV-induced lethality in a mouse model. As anticipated, mice in the JEV-infected vehicle-treated group started to show symptoms, including limb paralysis, restriction of movement, piloerection, body stiffening, and whole-body tremor, from day 5 postinfection. Within 21 days postinfection, most mice in the JEV-infected group succumbed to the infection, with the mortality rate being 73% 4 out of 15 animals survived . Manidipine treatment following JEV infection reduced the mortality rate to 20% 12 out of 15 animals survived Fig. 7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group.", "7A . Mice treated with manidipine alone or treated with manidipine and infected with JEV showed little abnormal behavior, similar to the findings for the mice in the vehicle-treated group. These results suggest that manidipine provided effective protection against JEVinduced mortality. To further relate these protective effects to the viral load and histopathological changes in the mouse brains, the viral titer was determined and mouse brain sections were collected and assayed at day 5 and day 21 postinfection, since mice started to show symptoms of JEV infection from day 5 postinfection and most of the surviving mice had recovered at day 21. The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B .", "The results indicated that, during the progression of the disease, manidipine treatment significantly reduced the viral load in infected mice compared to that in infected mice not receiving treatment, while no plaques formed in either the manidipine-or vehicle-treated group, and viral loads were undetectable in each group on day 21 postinfection Fig. 7B . As JEV was rapidly cleared from the blood after inoculation and was present in the lymphatic system during the preclinical phase, the effects of manidipine on infection of serum and the spleen were evaluated at earlier time points to detect whether the drug reduced the peripheral viral loads . . As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain.", "As shown in Fig. 7C , manidipine had little effect on peripheral JEV infection, which indicated that manidipine protected the mice against JEV-induced lethality by decreasing the viral load in the brain. Similarly, apparent damage in the brain, including meningitis, perivascular cuffing, vacuolar degeneration, and glial nodules, was observed in the JEV-infected and vehicle-treated group on day 5 postinfection, while manidipine treatment remarkably alleviated these phenomena Fig. 7D . These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors.", "These results indicate that the alleviation of histopathological changes was accompanied by a reduction in the viral load as well as a reduction in the rate of mortality, further confirming the curative effects of manidipine on viral encephalitis. Among the five hit drugs, manidipine, cilnidipine, and benidipine hydrochloride were VGCC inhibitors. It has been well documented in the literature that Ca 2ϩ inhibitors serve to inhibit virus infection at the stage of either entry . or replication . and even at the stage of budding . .", "or replication . and even at the stage of budding . . To this end, we first reviewed all 21 calcium inhibitors included in the current library of FDA-approved drugs and found that, in addition to the four DHP VGCC inhibitors listed in Fig. 1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen.", "1B , two other calcium inhibitors, i.e., flunarizine dihydrochloride and lomerizine hydrochloride, were also identified to be primary candidates with levels of inhibition of Ͼ90%. Similarly, three calcium channel antagonists, nisoldipine, felodipine, and nicardipine hydrochloride, showed levels of inhibition of 75%, 72%, and 66%, respectively, in the primary screen. Together, 9 of the 21 calcium inhibitors in the library, accounting for nearly half of the calcium inhibitors, exhibited levels of flavivirus inhibition of greater than 50%, suggesting that calcium, especially the calcium channel, is a potential antiviral target. To address this, another type of VGCC inhibitor, verapamil, an FDA-approved drug not yet included in the drug library used in this study, was investigated. Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection.", "Likewise, a Ca 2ϩ chelator, BAPTA-AM, as well as the Ca 2ϩ inhibitors 2-APB and cyclosporine, targeting ER and the mitochondrial Ca 2ϩ channel, respectively, were employed to investigate the response of JEV infection to the decrease in intracellular Ca 2ϩ levels. In line with the activities of the three hit DHP VGCC inhibitor drugs, the additional Ca 2ϩ inhibitors exerted anti-JEV activity, which indicated that Ca 2ϩ is indispensable for JEV infection. Thus, Ca 2ϩ inhibitors might be utilized as effective treatments for flavivirus infection. As the hit drugs exerted full inhibitory activity when they were added posttreatment, we believe that Ca 2ϩ is important for flavivirus genome replication. Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane .", "Furthermore, selection and genetic analysis of drug-resistant viruses revealed that NS4B is the viral target of manidipine. NS4B is part of the viral replication complex and is supposed to anchor the viral replicase to the ER membrane . . Meanwhile, the N-terminal 125amino-acid domain of DENV NS4B was indicated to be responsible for inhibition of the immune response . . Notably, several structurally distinct compounds have been identified to inhibit flavivirus replication by intensively targeting the TMD of NS4B . . . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication.", ". . . . . . . It is thus conceivable that inhibitors targeting TMD of NS4B would perturb its function, leading to the suppression of viral RNA replication. In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility.", "In this study, the replacement of Q130 of NS4B with a basic amino acid conferred the resistance effect without suppressing JEV replication, suggesting that position 130 could tolerate a basic amino acid and that the basic amino acid might be involved in the interplay of NS4B with host proteins rather than viral proteins. Moreover, the efficacy and toxicity of manidipine were monitored in vivo, with manidipine demonstrating effective antiviral activity with favorable biocompatibility. However, the dose used in this study was higher than the dose typically used clinically, representing one of the scenarios most commonly encountered in drug repurposing . . As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe.", "As manidipine was approved for use for the long-term treatment of hypertension . , pulse-dose treatment with manidipine over the shorter period of time required for the treatment of virus infection might be relatively safe. Moreover, use of a combination of manidipine with other Ca 2ϩ inhibitors might improve its therapeutic efficacy, reduce its toxicity, and reduce the risk of resistance development . . . . Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2.", ". Besides the three VGCC inhibitors, two hit drugs, pimecrolimus and nelfinavir mesylate, showed equivalent inhibitory activities on the replication of JEV, ZIKV, WNV, and DENV-2. Although there has been no report on the use of pimecrolimus for the treatment of infectious diseases, we showed that it had a robust effect against JEV with an SI of Ͼ32. The maximum plasma concentration C max of nelfinavir mesylate achieved with an adult dose was 3 to 4 g/ml . , which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease .", ", which was comparable to the IC 50 reported here. Notably, nelfinavir mesylate was confirmed to inhibit herpes simplex virus 1 HSV-1 and the replication of several other herpesviruses by interfering directly or indirectly with the later steps of virus formation, such as glycoprotein maturation or virion release, other than functioning in herpesviruses protease . . Whether nelfinavir mesylate inhibits flavivirus by interference with the virus protease or by other off-target effects is unknown. Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses.", "Understanding of the mechanism of the antiflavivirus effects of these drugs might uncover novel targets of the drugs, providing further insight into the pathogenesis of flaviviruses. Above all, the findings reported here provide novel insights into the molecular mechanisms underlying flavivirus infection and offer new and promising therapeutic possibilities for combating infections caused by flaviviruses. Cells and viruses. BHK-21, SH-SY5Y human neuroblastoma , Vero, and Huh-7 cells were cultured in Dulbecco modified Eagle medium HyClone, Logan, UT, USA supplemented with 10% fetal bovine serum Gibco, Grand Island, NY, USA . JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described .", "JEV strain AT31, the WNV replicon, and the DENV-2 replicon expressing Renilla luciferase Rluc were kindly provided by Bo Zhang, Wuhan Institute of Virology, Chinese Academy of Sciences CAS , China. JEV replicon recombinant viral particles RVPs were generated as previously described . . ZIKV strain H/PF/2013, kindly provided by the European Virus Archive Goes Global, was propagated and titrated in Vero cells. Optimization of HTS assay conditions. The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well .", "The cell density and RVP dose were optimized for the HTS assay. Vero cells at different densities 2,500 to 12,500 cells per well were infected with from 1.25 to 20 l RVPs 1 to 16 copies per well . The appropriate cell density as well as the RVP dose was selected by comparing the S/B ratio, CV, and Z= values under different conditions as previously described . . Methyl-␤-cyclodextrin and dimethyl sulfoxide DMSO were used as positive and negative controls, respectively. HTS assay of an FDA-approved compound library. A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use.", "A library of 1,018 FDA-approved drugs was purchased from Selleck Chemicals Houston, TX, USA . The compounds were stored as 10 mM stock solutions in DMSO at 4°C until use. The first round of the HTS assay was carried out as shown in Fig. 1A . The criteria used to identify the primary candidates were no apparent cytotoxicity and an average level of inhibition of Ͼ90% in duplicate wells. The criteria of dose-dependent inhibition and cell viability of Ͼ80% were applied for the reconfirmation screen. Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs.", "Furthermore, the CC 50 of each compound was calculated, and those compounds displaying SIs over 10 were considered hits in this study. Identification of antiviral effects of five hit drugs. The antiviral effects of the drugs were evaluated by quantitative reverse transcription-PCR qRT-PCR , immunofluorescence assay IFA , and plaque assay as previously reported . . . . . The experimental timeline is depicted in Fig. 2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively.", "2A . To ensure the effectiveness of the hit drugs in flavivirus replication, BHK-21 cells transfected with the JEV, WNV, or DENV-2 replicon were incubated with each drug at the concentrations indicated above, and the luciferase activities were determined 24 h, 48 h, or 72 h later, respectively. Time-of-addition experiment. To evaluate which stage of the JEV life cycle was inhibited by each hit, a time-of-addition experiment was performed as previously described . . Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h .", ". Vero cells were infected with 20 l RVPs for 1 h 0 to 1 h . The test compounds were incubated with the cells for 1 h before infection Ϫ1 to 0 h , during infection 0 to 1 h , and for 23 h postinfection 1 to 24 h Fig. 3A . To exclude a possible direct inactivating effect of the drugs, RVPs were incubated with each drug at 37°C for 1 h, and the mixtures were diluted 25-fold to infect Vero cells. Twenty-four hours later, the luciferase activities were determined as described above Fig. 3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine.", "3A . Manidipine-resistant virus. Manidipine-resistant virus was generated by passaging of JEV on Vero cells in the presence of manidipine. Passages 1 to 10 used 5 M manidipine, and passages 11 to 20 used 10 M manidipine. As a control, WT virus was passaged in the presence of 2% DMSO in parallel. Passaging was terminated at passage 20, when no further improvement in resistance was detected. Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing.", "Two manidipine-resistant virus isolates were plaque purified and amplified in the presence of manidipine. Viral RNA was extracted, amplified, and purified for sequencing. An infectious cDNA clone of JEV, strain AT31 pMWJEAT , kindly provided by T. Wakita, Tokyo Metropolitan Institute for Neuroscience, was used to recover WT and mutant viruses as described previously . . Virus titers and manidipine sensitivities were determined by plaque assay in Vero cells. Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China .", "Manidipine administration to JEV-infected mice. Adult female BALB/c mice age, 4 weeks were kept in the Laboratory Animal Center of Wuhan Institute of Virology, CAS Wuhan, China . The mice were randomly divided into four groups 30 mice per group : a JEV-infected and vehicle 2% Tween 80 plus 5% DMSO in phosphate-buffered saline PBS -treated group, a manidipine-treated group, a JEV-infected and manidipine-treated group, and a vehicle-treated group. For infection, mice were infected intraperitoneally with 5 ϫ 10 6 PFU of JEV strain AT31. For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days.", "For the manidipine and vehicle treatments, mice were injected intraperitoneally with 25 mg/kg of body weight manidipine or PBS with 2% Tween 80 and 5% DMSO, respectively. Treatments were administered twice a day for the first 2 days and then consecutively administered once a day for up to 21 days. Five mice from each group were sacrificed on days 1, 3, and 5 postinfection. Serum, spleen tissue, and brain tissue samples were collected for viral titer determination and histopathology investigation. Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China.", "Fifteen mice were monitored daily for morbidity and mortality. The mice that showed neurological signs of disease were euthanized according to the Regulations for the Administration of Affairs Concerning Experimental Animals in China. The protocols were reviewed and approved by the Laboratory Animal Care and Use Committee at the Wuhan Institute of Virology, CAS Wuhan, China ." ]

[ "The vacuolar-type H + -ATPase v-ATPase is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells ECs , in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process.", "The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.", "Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound. Text: The vacuolar-type H + -ATPase v-ATPase is the major proton pump responsible for acidification of intracellular compartments in eukaryotic cells . The enzyme consists of two multi-subunit complexes, the soluble V 1 transmembrane V o subcomplex required for the proton transport across membranes . In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing.", "In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing. In specialized cell types including osteoclasts and renal epithelial cells, v-ATPases can also be expressed on the plasma membrane, where they pump protons into the extracellular space . In cancer cells v-ATPases are expressed on the plasma membrane in order to eliminate toxic cytosolic H + . Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years.", "Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years. Archazolid A and B are highly potent and specific inhibitors of v-ATPases . They were first isolated from the myxobacterium Archangium gephyra . These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development.", "These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development. Archazolids can be either produced by fermentation or by total synthesis . In the field of cancer research several studies reported on interesting pharmacological effects of archazolid: It reduced the migration of different invasive tumor cells in vitro and cancer cell metastasis in vivo in a breast tumor mouse model . Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression .", "Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression . Up to now, the role of v-ATPases in endothelial cells has only rarely been investigated. The endothelium plays a crucial role in the pathogenesis and progression of cancer: The metastatic cascade includes local angiogenesis at the site of the primary tumor and adhesion of tumor cells at the site of metastasis . Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth .", "Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth . Circulating cancer cells can adhere to the endothelium at distant sites. This adhesive interaction is mediated by receptors and corresponding ligands expressed on tumor and endothelial cells . V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells .", "V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells . So far, there are no investigations on the role of endothelial v-ATPases for the process of tumor cell adhesion onto the endothelium. Thus, we were interested in the consequences of the inhibition of endothelial v-ATPase by archazolid on the interaction between endothelial and cancer cells. Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins.", "Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins. For the first time, our study revealed a link between the function of v-ATPases and the adhesion and transmigration properties of endothelial cells. CellTiter-Blue Cell Viability Assay Promega, Mannheim, Germany was performed according to the manufacturer's protocol for determining the cell viability of cells after treatment with archazolid. This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment.", "This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment. Fluorescence was measured with an Infinite F200 pro microplate reader Tecan, Männedorf, Switzerland at 560 nm excitation and 590 nm emission . CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega was performed according to the manufacturer's instructions for determining the lactate dehydrogenase LDH release after treatment with archazolid. Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells.", "Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells. Released LDH catalyzes the enzymatic conversion of lactate to pyruvate which provides NADH for the conversion of iodonitrotetrazolium violet into a red formazan product in the presence of diaphorase. The absorbance was measured with a Varioskan Flash microplate reader Thermo Fisher Scientific at 490 nm. LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes.", "LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes. Both dyes were incubated for 10 min at 37˚C before acquisition of single images by a Leica DMI6000 B fluorescence microscope Leica Microsystems, Wetzlar, Germany . HUVECs were seeded in collagen G-coated 24-well plates and grown to confluence for two days before treatment. The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ .", "The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ . Tumor cell adhesion was determined by fluorescence measurements with an Infinite F200 pro microplate reader Tecan at 485 nm excitation and 535 nm emission . For blocking the integrin β1 subunit on MDA-MB-231 or PC-3 cells, CellTracker Greenlabeled MDA-MB-231 or PC-3 cells were incubated with an anti-integrin β1 antibody P5D2, ab24693, Abcam, Cambridge, United Kingdom at a concentration of 1 μg antibody per one million cells in 1 ml DMEM. Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM .", "Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM . HUVECs were washed once with ECGM before untreated MDA-MB-231 or PC-3 cells were added to HUVECs. For the adhesion of MDA-MB-231 or PC-3 cells onto extracellular matrix ECM components 24-well plates were coated with collagen G 10 μg/ml in PBS , human plasma fibronectin 10 μg/ml PBS or laminin-411 10 μg/ml in Dulbecco's PBS DPBS containing Ca 2+ and Mg 2+ at 4˚C overnight. The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated.", "The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated. Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye as described in the section cell adhesion assay and resuspended in medium 199 PAN-Biotech containing 0.1% BSA. HUVECs were washed twice with medium 199 containing 0.1% BSA before MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Medium 199 containing 0.1% BSA was used as negative control and medium 199 containing 20% FCS was used as chemoattractant for transmigration in the lower compartment. Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission .", "Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission . HUVECs were grown to confluence on 6-well plates before they were treated with archazolid for 12 h. The cells were induced to upregulate the gene expression of cell adhesion molecules by TNFα. RNA was isolated using the RNeasy Mini Kit from Qiagen Hilden, Germany according to the manufacturer's protocol. On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific .", "On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific . qPCR experiments were performed using a StepOnePlus System Applied Biosystems, Thermo Fisher Scientific and data was analyzed by the StepOne and Ste-pOnePlus Software v2.3. Power SYBR Green PCR Master Mix Life Technologies and the comparative C T quantitation method 2 -ΔΔCT were used. HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany .", "HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany . In the case of ICAM-1 the detached cells were fixed with 4% formaldehyde Polysciences, Hirschberg an der Bergstraße, Germany in PBS for 10 min and washed once with PBS before incubating with the fluorescein isothiocyanate FITC -labeled anti-human CD54 mouse, ICAM-1 antibody MCA1615F, Biozol, Eching, Germany at room temperature for 45 min. For all other proteins, the cells were not fixed and washed once with PBS before incubating with the antibodies phycoerythrin PE -labeled anti-human CD106 mouse, VCAM-1 , PE-labeled anti-human CD62E mouse, E-selectin and PE-labeled anti-human CD325 mouse, N-cadherin from Becton Dickinson on ice for 45 min. These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany .", "These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany . To stain the surface collagen on HUVECs, cells were incubated with an anti-human collagen antibody rabbit, 1:40, ab36064, Abcam on ice for 30 min. The staining procedure was performed on ice to ensure that surface proteins or antibodies are not endocytosed. The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei.", "The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei. Confluent HUVECs in 6-well plates were treated as indicated. Cells were washed with ice-cold PBS and lysed with RIPA buffer supplemented with protease inhibitors Complete Mini EDTA-free; Roche, Mannheim, Germany , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride, β-glycerophosphate, sodium pyrophosphate and H 2 O 2 . Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany .", "Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany . Separated proteins were transferred onto polyvinylidene difluoride membranes by tank blotting Bio-Rad Laboratories for immunodetection. Membranes were blocked with 5% boltinggrade milk powder Carl Roth in TBS containing 0.1% Tween 20 Sigma-Aldrich . The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis.", "The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis. Cathepsin B activity assay was performed as described in the publication by Kubisch et al. . Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride.", "Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride. The fluorogenic cathepsin B substrate Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride C5429, Sigma-Aldrich was added to 30 μg of the cell lysate diluted in assay buffer supplemented with 2 mM L-cysteine C7880, Sigma-Aldrich and incubated for 30 min at 40˚C. Fluorescence was measured at 348 nm excitation and 440 nm emission with a microplate reader Varioskan Flash, Thermo Fisher Scientific . The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction.", "The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction. The HUVEC Nucleofector Kit Lonza, Cologne, Germany was used to transfect HUVECs. The transfection was performed according to the manufacturer's protocol using 2.5 μg plasmid DNA for 500,000 cells Nucleofector 2b Device, Lonza . 48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe .", "48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe . hCathepsin B was digested with PmeI and XbaI and the linear DNA fragment not corresponding to the human CTSB gene was religated to generate the empty pcDNA3.1 - delta MCS plasmid that was used for control transfections. The original backbone of hCathepsin B is the pcDNA3.1 - from Thermo Fisher Scientific. The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA .", "The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA . One-way ANOVA followed by Tukey's post-hoc test or unpaired t-test was used for the evaluation of a minimum of three independent experiments. The numbers of independently performed experiments n are stated in the corresponding figure legends. p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM .", "p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM . Since the v-ATPase inhibitor archazolid has never been used for studies in endothelial cells, we first performed cytotoxicity assays. We treated confluent HUVECs with up to 1 nM archazolid for 24 and 48 h and observed that this treatment has neither an influence on the metabolic activity nor on the release of LDH after 24 h Fig 1A and 1B, left panels . The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings.", "The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings. Microscopic analysis revealed that also the integrity of the endothelial monolayer was not affected by archazolid, but the cells showed a slightly different morphology Fig 2A : Archazolid-treated cells were swollen compared to control cells, which was not unexpected, as vacuolation of the endoplasmic reticulum ER has been described for other cell types and is typical for v-ATPase inhibitors . This effect was obvious both in subconfluent and in confluent cells Fig 2A . Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells.", "Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells. Thus, this dye can serve as an indicator of v-ATPase inhibition. To proof that archazolid is also functionally active as a v-ATPase inhibitor in HUVECs, cells were treated with 1 nM archazolid before they were incubated with LysoTracker Red DND-99 and Hoechst 33342. As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase.", "As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase. We analyzed the adhesion of MDA-MB-231 cells onto HUVECs. Confluent HUVECs were treated with up to 1 nM archazolid for 24 h. Untreated MDA-MB-231 cells were labeled with Cell-Tracker Green CMFDA Dye. Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells.", "Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells. HUVECs seeded in a Boyden chamber were treated with 1 nM archazolid for 24 h. CellTracker Green-labeled MDA-MB-231 cells not treated with archazolid were allowed to transmigrate through the endothelial monolayer for 24 h. As shown in Fig 3C, archazolid significantly decreased the transendothelial migration of MDA-MB-231 cells. The influence of archazolid on tumor cell adhesion was not only studied in HUVECs, which represent macrovascular endothelial cells, but also in microvascular HMEC-1 cells. Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B .", "Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B . Of note, the adhesion of non-metastatic Jurkat cells, an acute T cell leukemia cell line, remained unaffected after treatment of HUVECs with archazolid S1A Fig . Taken together, archazolid treatment augmented the adhesive properties of both micro-and macrovascular endothelial cells for metastatic tumor cells, but not for non-metastatic ones. Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process.", "Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process. Ligands for the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin were found to be expressed on tumor cells and to mediate tumor-endothelial cell interaction . Inhibition of the v-ATPase might affect the expression of endothelial cell adhesion molecules on mRNA or protein levels. To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A .", "To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A . The protein expression of these adhesion molecules on the surface of endothelial cells was analyzed by flow cytometry. Archazolid 1 nM, 24 h did not affect the cell surface expression of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5B . Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners.", "Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners. Within this protein family β1-integrins have been reported to mediate tumor cell adhesion onto quiescent endothelial cells . In order to elucidate the role of β1-integrins for the archazolid-induced tumor cell adhesion, the integrin β1-subunit was blocked either on MDA-MB-231 cells, PC-3 cells or on HUVECs. Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels .", "Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels . Depending on their α subunit, β1-integrins have a variety of ligands including extracellular matrix ECM components such as collagen, fibronectin and laminin . Therefore, we hypothesized that archazolid treatment of endothelial cells might lead to an upregulation of these components. MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A .", "MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A . MDA-MB-231 and PC-3 cells also adhered to fibronectin-coated plastic, but to a much lesser extent compared to the collagen coating. Therefore, we focused on the interaction between these two tumor cell lines and collagen. Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix.", "Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix. To detect collagen on the endothelial surface, archazolid-treated HUVECs were labeled with an antibody against collagen type I-IV on ice to prevent endocytosis and to ensure that the antibody does not bind to intracellular collagen. Interestingly, archazolid increased the amount of surface collagen on HUVECs by about 50% Fig 7C . Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen .", "Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen . As collagen is degraded by cathepsin B and the activation of cathepsin B depends on v-ATPase activity 28, 42 , we suggested that an accumulation of collagen on the surface of endothelial cells might be a consequence of an impaired functionality of cathepsin B. Therefore, an enzyme activity assay based on the proteolysis of a fluorogenic cathepsin B substrate was performed. In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A .", "In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A . In line with this result, western blot analysis showed that archazolid 1 nM reduces the protein expression of the mature, active form of cathepsin B to less than 40% of the control in HUVECs Fig 8B . To proof whether the archazolid-induced tumor cell adhesion is a consequence of the decreased amount of cathepsin B, HUVECs were transfected with a plasmid coding for human cathepsin B or with the empty vector as control. After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A .", "After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A . Overexpression of cathepsin B strongly diminished both the basal and the archazolid-induced adhesion of MDA-MB-231 cells Fig 9B . Targeting the proton pump v-ATPase for cancer therapy has gained great interest since its inhibition was reported to reduce the invasiveness of cancer cells and, most importantly, also metastasis . Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions.", "Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions. For the first time, we were able to show a link between v-ATPase and the adhesion and transmigration properties of the endothelium. Inhibition of the v-ATPase in endothelial cells by archazolid significantly increased the adhesion of metastatic cancer cells and decreased the transendothelial migration of cancer cells which was attributed to augmented collagen levels on the surface on archazolid-treated endothelial cells. Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound.", "Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound. However, we found that the archazolid-induced tumor cell adhesion was mediated by β1-integrins expressed on MDA-MB-231 breast cancer and PC-3 prostate cancer cells as blocking of the integrin β1 subunit on these tumor cells reversed the pro-adhesive effect of archazolid. In adhesion experiments on plastic coated with extracellular matrix components, we could show that MDA-MB-231 and PC-3 cells clearly favored the interaction with collagen, whereas the adhesion of non-metastatic Jurkat cells was largely independent from extracellular matrix proteins S1B Fig . The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins .", "The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins . α4β1integrins are receptors for VCAM-1 and fibronectin and it has been shown that Jurkat cells interact with human endothelial cells that express VCAM-1 after cytokine treatment or cells transfected with VCAM-1 . Our results are in line with previous studies showing that α2β1-and α3β1-integrin expressing MDA-MB-231 and PC-3 cells were able to rapidly attach to collagen in the cortical bone matrix. In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin .", "In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin . Though expressing receptors for fibronectin and laminin, MDA-MB-231 and PC-3 cells adhered to fibronectin to a much lesser extent and did not adhere to laminin, probably due to lower affinities to these extracellular matrix components. Importantly, v-ATPase inhibition by archazolid increased the surface levels of the extracellular matrix component collagen, which might explain that the increase of MDA-MB-231 and PC-3 cells onto archazolid-treated HUVECs is independent of endothelial cell adhesion molecules. By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation .", "By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation . In addition, recent studies reported that v-ATPase inhibition impairs the activity of cathepsin B in cancer cells . Therefore, we suggested that the accumulation of collagen on the endothelial surface might be a consequence of impaired cathepsin B activity or expression in endothelial cells. In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes.", "In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes. Following transport to the Golgi apparatus, the preprocathepsin B is glycosylated with mannose-containing oligosaccharides. The targeting of procathepsin B to lysosomes is mannose-6-phosphate receptor-dependent and its dissociation from the receptor as well as its proteolytic processing into mature cathepsin B requires acidification of the compartment . In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism.", "In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism. Interestingly, overexpression of cathepsin B attenuated the archazolid-induced adhesion of breast cancer cells onto endothelial cells, indicating that the adhesion negatively correlates with the expression of cathepsin B. As cathepsin B can also degrade other extracellular matrix components such as fibronectin and laminin , v-ATPase inhibition could lead to an accumulation of these proteins and an increased adhesion of cells expressing fibronectin or laminin receptors. However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig .", "However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig . Interestingly . In hepatic cancer cells, archazolid reduces Ras/Raf/MEK/ERK signaling by altering the membrane composition and fluidity . We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells.", "We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells. As a result, the amount of collagen on the surface of endothelial cells was significantly upregulated, which finally resulted in an increased adhesion of the β1-integrin-expressing metastatic cancer cell lines MDA-MB-231 and PC-3 onto archazolidtreated endothelial cells, whereas the adhesion of non-metastatic Jurkat cells was unaffected. This study shows that the v-ATPase plays an important role in regulating the adhesion of cells expressing receptors for extracellular matrix components. Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells.", "Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells. The fact that the adhesion of metastatic tumor cells onto endothelial cells is increased while their transendothelial migration is reduced upon inhibition of endothelial v-ATPase by archazolid further supports the view of archazolid as a potential anti-metastatic compound." ]

[ "The vacuolar-type H + -ATPase v-ATPase is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells ECs , in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process.", "The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.", "Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound. Text: The vacuolar-type H + -ATPase v-ATPase is the major proton pump responsible for acidification of intracellular compartments in eukaryotic cells . The enzyme consists of two multi-subunit complexes, the soluble V 1 transmembrane V o subcomplex required for the proton transport across membranes . In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing.", "In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing. In specialized cell types including osteoclasts and renal epithelial cells, v-ATPases can also be expressed on the plasma membrane, where they pump protons into the extracellular space . In cancer cells v-ATPases are expressed on the plasma membrane in order to eliminate toxic cytosolic H + . Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years.", "Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years. Archazolid A and B are highly potent and specific inhibitors of v-ATPases . They were first isolated from the myxobacterium Archangium gephyra . These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development.", "These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development. Archazolids can be either produced by fermentation or by total synthesis . In the field of cancer research several studies reported on interesting pharmacological effects of archazolid: It reduced the migration of different invasive tumor cells in vitro and cancer cell metastasis in vivo in a breast tumor mouse model . Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression .", "Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression . Up to now, the role of v-ATPases in endothelial cells has only rarely been investigated. The endothelium plays a crucial role in the pathogenesis and progression of cancer: The metastatic cascade includes local angiogenesis at the site of the primary tumor and adhesion of tumor cells at the site of metastasis . Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth .", "Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth . Circulating cancer cells can adhere to the endothelium at distant sites. This adhesive interaction is mediated by receptors and corresponding ligands expressed on tumor and endothelial cells . V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells .", "V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells . So far, there are no investigations on the role of endothelial v-ATPases for the process of tumor cell adhesion onto the endothelium. Thus, we were interested in the consequences of the inhibition of endothelial v-ATPase by archazolid on the interaction between endothelial and cancer cells. Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins.", "Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins. For the first time, our study revealed a link between the function of v-ATPases and the adhesion and transmigration properties of endothelial cells. CellTiter-Blue Cell Viability Assay Promega, Mannheim, Germany was performed according to the manufacturer's protocol for determining the cell viability of cells after treatment with archazolid. This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment.", "This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment. Fluorescence was measured with an Infinite F200 pro microplate reader Tecan, Männedorf, Switzerland at 560 nm excitation and 590 nm emission . CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega was performed according to the manufacturer's instructions for determining the lactate dehydrogenase LDH release after treatment with archazolid. Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells.", "Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells. Released LDH catalyzes the enzymatic conversion of lactate to pyruvate which provides NADH for the conversion of iodonitrotetrazolium violet into a red formazan product in the presence of diaphorase. The absorbance was measured with a Varioskan Flash microplate reader Thermo Fisher Scientific at 490 nm. LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes.", "LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes. Both dyes were incubated for 10 min at 37˚C before acquisition of single images by a Leica DMI6000 B fluorescence microscope Leica Microsystems, Wetzlar, Germany . HUVECs were seeded in collagen G-coated 24-well plates and grown to confluence for two days before treatment. The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ .", "The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ . Tumor cell adhesion was determined by fluorescence measurements with an Infinite F200 pro microplate reader Tecan at 485 nm excitation and 535 nm emission . For blocking the integrin β1 subunit on MDA-MB-231 or PC-3 cells, CellTracker Greenlabeled MDA-MB-231 or PC-3 cells were incubated with an anti-integrin β1 antibody P5D2, ab24693, Abcam, Cambridge, United Kingdom at a concentration of 1 μg antibody per one million cells in 1 ml DMEM. Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM .", "Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM . HUVECs were washed once with ECGM before untreated MDA-MB-231 or PC-3 cells were added to HUVECs. For the adhesion of MDA-MB-231 or PC-3 cells onto extracellular matrix ECM components 24-well plates were coated with collagen G 10 μg/ml in PBS , human plasma fibronectin 10 μg/ml PBS or laminin-411 10 μg/ml in Dulbecco's PBS DPBS containing Ca 2+ and Mg 2+ at 4˚C overnight. The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated.", "The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated. Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye as described in the section cell adhesion assay and resuspended in medium 199 PAN-Biotech containing 0.1% BSA. HUVECs were washed twice with medium 199 containing 0.1% BSA before MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Medium 199 containing 0.1% BSA was used as negative control and medium 199 containing 20% FCS was used as chemoattractant for transmigration in the lower compartment. Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission .", "Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission . HUVECs were grown to confluence on 6-well plates before they were treated with archazolid for 12 h. The cells were induced to upregulate the gene expression of cell adhesion molecules by TNFα. RNA was isolated using the RNeasy Mini Kit from Qiagen Hilden, Germany according to the manufacturer's protocol. On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific .", "On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific . qPCR experiments were performed using a StepOnePlus System Applied Biosystems, Thermo Fisher Scientific and data was analyzed by the StepOne and Ste-pOnePlus Software v2.3. Power SYBR Green PCR Master Mix Life Technologies and the comparative C T quantitation method 2 -ΔΔCT were used. HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany .", "HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany . In the case of ICAM-1 the detached cells were fixed with 4% formaldehyde Polysciences, Hirschberg an der Bergstraße, Germany in PBS for 10 min and washed once with PBS before incubating with the fluorescein isothiocyanate FITC -labeled anti-human CD54 mouse, ICAM-1 antibody MCA1615F, Biozol, Eching, Germany at room temperature for 45 min. For all other proteins, the cells were not fixed and washed once with PBS before incubating with the antibodies phycoerythrin PE -labeled anti-human CD106 mouse, VCAM-1 , PE-labeled anti-human CD62E mouse, E-selectin and PE-labeled anti-human CD325 mouse, N-cadherin from Becton Dickinson on ice for 45 min. These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany .", "These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany . To stain the surface collagen on HUVECs, cells were incubated with an anti-human collagen antibody rabbit, 1:40, ab36064, Abcam on ice for 30 min. The staining procedure was performed on ice to ensure that surface proteins or antibodies are not endocytosed. The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei.", "The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei. Confluent HUVECs in 6-well plates were treated as indicated. Cells were washed with ice-cold PBS and lysed with RIPA buffer supplemented with protease inhibitors Complete Mini EDTA-free; Roche, Mannheim, Germany , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride, β-glycerophosphate, sodium pyrophosphate and H 2 O 2 . Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany .", "Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany . Separated proteins were transferred onto polyvinylidene difluoride membranes by tank blotting Bio-Rad Laboratories for immunodetection. Membranes were blocked with 5% boltinggrade milk powder Carl Roth in TBS containing 0.1% Tween 20 Sigma-Aldrich . The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis.", "The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis. Cathepsin B activity assay was performed as described in the publication by Kubisch et al. . Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride.", "Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride. The fluorogenic cathepsin B substrate Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride C5429, Sigma-Aldrich was added to 30 μg of the cell lysate diluted in assay buffer supplemented with 2 mM L-cysteine C7880, Sigma-Aldrich and incubated for 30 min at 40˚C. Fluorescence was measured at 348 nm excitation and 440 nm emission with a microplate reader Varioskan Flash, Thermo Fisher Scientific . The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction.", "The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction. The HUVEC Nucleofector Kit Lonza, Cologne, Germany was used to transfect HUVECs. The transfection was performed according to the manufacturer's protocol using 2.5 μg plasmid DNA for 500,000 cells Nucleofector 2b Device, Lonza . 48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe .", "48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe . hCathepsin B was digested with PmeI and XbaI and the linear DNA fragment not corresponding to the human CTSB gene was religated to generate the empty pcDNA3.1 - delta MCS plasmid that was used for control transfections. The original backbone of hCathepsin B is the pcDNA3.1 - from Thermo Fisher Scientific. The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA .", "The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA . One-way ANOVA followed by Tukey's post-hoc test or unpaired t-test was used for the evaluation of a minimum of three independent experiments. The numbers of independently performed experiments n are stated in the corresponding figure legends. p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM .", "p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM . Since the v-ATPase inhibitor archazolid has never been used for studies in endothelial cells, we first performed cytotoxicity assays. We treated confluent HUVECs with up to 1 nM archazolid for 24 and 48 h and observed that this treatment has neither an influence on the metabolic activity nor on the release of LDH after 24 h Fig 1A and 1B, left panels . The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings.", "The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings. Microscopic analysis revealed that also the integrity of the endothelial monolayer was not affected by archazolid, but the cells showed a slightly different morphology Fig 2A : Archazolid-treated cells were swollen compared to control cells, which was not unexpected, as vacuolation of the endoplasmic reticulum ER has been described for other cell types and is typical for v-ATPase inhibitors . This effect was obvious both in subconfluent and in confluent cells Fig 2A . Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells.", "Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells. Thus, this dye can serve as an indicator of v-ATPase inhibition. To proof that archazolid is also functionally active as a v-ATPase inhibitor in HUVECs, cells were treated with 1 nM archazolid before they were incubated with LysoTracker Red DND-99 and Hoechst 33342. As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase.", "As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase. We analyzed the adhesion of MDA-MB-231 cells onto HUVECs. Confluent HUVECs were treated with up to 1 nM archazolid for 24 h. Untreated MDA-MB-231 cells were labeled with Cell-Tracker Green CMFDA Dye. Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells.", "Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells. HUVECs seeded in a Boyden chamber were treated with 1 nM archazolid for 24 h. CellTracker Green-labeled MDA-MB-231 cells not treated with archazolid were allowed to transmigrate through the endothelial monolayer for 24 h. As shown in Fig 3C, archazolid significantly decreased the transendothelial migration of MDA-MB-231 cells. The influence of archazolid on tumor cell adhesion was not only studied in HUVECs, which represent macrovascular endothelial cells, but also in microvascular HMEC-1 cells. Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B .", "Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B . Of note, the adhesion of non-metastatic Jurkat cells, an acute T cell leukemia cell line, remained unaffected after treatment of HUVECs with archazolid S1A Fig . Taken together, archazolid treatment augmented the adhesive properties of both micro-and macrovascular endothelial cells for metastatic tumor cells, but not for non-metastatic ones. Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process.", "Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process. Ligands for the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin were found to be expressed on tumor cells and to mediate tumor-endothelial cell interaction . Inhibition of the v-ATPase might affect the expression of endothelial cell adhesion molecules on mRNA or protein levels. To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A .", "To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A . The protein expression of these adhesion molecules on the surface of endothelial cells was analyzed by flow cytometry. Archazolid 1 nM, 24 h did not affect the cell surface expression of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5B . Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners.", "Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners. Within this protein family β1-integrins have been reported to mediate tumor cell adhesion onto quiescent endothelial cells . In order to elucidate the role of β1-integrins for the archazolid-induced tumor cell adhesion, the integrin β1-subunit was blocked either on MDA-MB-231 cells, PC-3 cells or on HUVECs. Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels .", "Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels . Depending on their α subunit, β1-integrins have a variety of ligands including extracellular matrix ECM components such as collagen, fibronectin and laminin . Therefore, we hypothesized that archazolid treatment of endothelial cells might lead to an upregulation of these components. MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A .", "MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A . MDA-MB-231 and PC-3 cells also adhered to fibronectin-coated plastic, but to a much lesser extent compared to the collagen coating. Therefore, we focused on the interaction between these two tumor cell lines and collagen. Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix.", "Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix. To detect collagen on the endothelial surface, archazolid-treated HUVECs were labeled with an antibody against collagen type I-IV on ice to prevent endocytosis and to ensure that the antibody does not bind to intracellular collagen. Interestingly, archazolid increased the amount of surface collagen on HUVECs by about 50% Fig 7C . Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen .", "Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen . As collagen is degraded by cathepsin B and the activation of cathepsin B depends on v-ATPase activity 28, 42 , we suggested that an accumulation of collagen on the surface of endothelial cells might be a consequence of an impaired functionality of cathepsin B. Therefore, an enzyme activity assay based on the proteolysis of a fluorogenic cathepsin B substrate was performed. In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A .", "In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A . In line with this result, western blot analysis showed that archazolid 1 nM reduces the protein expression of the mature, active form of cathepsin B to less than 40% of the control in HUVECs Fig 8B . To proof whether the archazolid-induced tumor cell adhesion is a consequence of the decreased amount of cathepsin B, HUVECs were transfected with a plasmid coding for human cathepsin B or with the empty vector as control. After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A .", "After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A . Overexpression of cathepsin B strongly diminished both the basal and the archazolid-induced adhesion of MDA-MB-231 cells Fig 9B . Targeting the proton pump v-ATPase for cancer therapy has gained great interest since its inhibition was reported to reduce the invasiveness of cancer cells and, most importantly, also metastasis . Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions.", "Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions. For the first time, we were able to show a link between v-ATPase and the adhesion and transmigration properties of the endothelium. Inhibition of the v-ATPase in endothelial cells by archazolid significantly increased the adhesion of metastatic cancer cells and decreased the transendothelial migration of cancer cells which was attributed to augmented collagen levels on the surface on archazolid-treated endothelial cells. Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound.", "Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound. However, we found that the archazolid-induced tumor cell adhesion was mediated by β1-integrins expressed on MDA-MB-231 breast cancer and PC-3 prostate cancer cells as blocking of the integrin β1 subunit on these tumor cells reversed the pro-adhesive effect of archazolid. In adhesion experiments on plastic coated with extracellular matrix components, we could show that MDA-MB-231 and PC-3 cells clearly favored the interaction with collagen, whereas the adhesion of non-metastatic Jurkat cells was largely independent from extracellular matrix proteins S1B Fig . The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins .", "The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins . α4β1integrins are receptors for VCAM-1 and fibronectin and it has been shown that Jurkat cells interact with human endothelial cells that express VCAM-1 after cytokine treatment or cells transfected with VCAM-1 . Our results are in line with previous studies showing that α2β1-and α3β1-integrin expressing MDA-MB-231 and PC-3 cells were able to rapidly attach to collagen in the cortical bone matrix. In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin .", "In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin . Though expressing receptors for fibronectin and laminin, MDA-MB-231 and PC-3 cells adhered to fibronectin to a much lesser extent and did not adhere to laminin, probably due to lower affinities to these extracellular matrix components. Importantly, v-ATPase inhibition by archazolid increased the surface levels of the extracellular matrix component collagen, which might explain that the increase of MDA-MB-231 and PC-3 cells onto archazolid-treated HUVECs is independent of endothelial cell adhesion molecules. By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation .", "By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation . In addition, recent studies reported that v-ATPase inhibition impairs the activity of cathepsin B in cancer cells . Therefore, we suggested that the accumulation of collagen on the endothelial surface might be a consequence of impaired cathepsin B activity or expression in endothelial cells. In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes.", "In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes. Following transport to the Golgi apparatus, the preprocathepsin B is glycosylated with mannose-containing oligosaccharides. The targeting of procathepsin B to lysosomes is mannose-6-phosphate receptor-dependent and its dissociation from the receptor as well as its proteolytic processing into mature cathepsin B requires acidification of the compartment . In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism.", "In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism. Interestingly, overexpression of cathepsin B attenuated the archazolid-induced adhesion of breast cancer cells onto endothelial cells, indicating that the adhesion negatively correlates with the expression of cathepsin B. As cathepsin B can also degrade other extracellular matrix components such as fibronectin and laminin , v-ATPase inhibition could lead to an accumulation of these proteins and an increased adhesion of cells expressing fibronectin or laminin receptors. However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig .", "However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig . Interestingly . In hepatic cancer cells, archazolid reduces Ras/Raf/MEK/ERK signaling by altering the membrane composition and fluidity . We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells.", "We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells. As a result, the amount of collagen on the surface of endothelial cells was significantly upregulated, which finally resulted in an increased adhesion of the β1-integrin-expressing metastatic cancer cell lines MDA-MB-231 and PC-3 onto archazolidtreated endothelial cells, whereas the adhesion of non-metastatic Jurkat cells was unaffected. This study shows that the v-ATPase plays an important role in regulating the adhesion of cells expressing receptors for extracellular matrix components. Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells.", "Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells. The fact that the adhesion of metastatic tumor cells onto endothelial cells is increased while their transendothelial migration is reduced upon inhibition of endothelial v-ATPase by archazolid further supports the view of archazolid as a potential anti-metastatic compound." ]

[ "The vacuolar-type H + -ATPase v-ATPase is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells ECs , in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process.", "The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.", "Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound. Text: The vacuolar-type H + -ATPase v-ATPase is the major proton pump responsible for acidification of intracellular compartments in eukaryotic cells . The enzyme consists of two multi-subunit complexes, the soluble V 1 transmembrane V o subcomplex required for the proton transport across membranes . In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing.", "In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing. In specialized cell types including osteoclasts and renal epithelial cells, v-ATPases can also be expressed on the plasma membrane, where they pump protons into the extracellular space . In cancer cells v-ATPases are expressed on the plasma membrane in order to eliminate toxic cytosolic H + . Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years.", "Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years. Archazolid A and B are highly potent and specific inhibitors of v-ATPases . They were first isolated from the myxobacterium Archangium gephyra . These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development.", "These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development. Archazolids can be either produced by fermentation or by total synthesis . In the field of cancer research several studies reported on interesting pharmacological effects of archazolid: It reduced the migration of different invasive tumor cells in vitro and cancer cell metastasis in vivo in a breast tumor mouse model . Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression .", "Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression . Up to now, the role of v-ATPases in endothelial cells has only rarely been investigated. The endothelium plays a crucial role in the pathogenesis and progression of cancer: The metastatic cascade includes local angiogenesis at the site of the primary tumor and adhesion of tumor cells at the site of metastasis . Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth .", "Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth . Circulating cancer cells can adhere to the endothelium at distant sites. This adhesive interaction is mediated by receptors and corresponding ligands expressed on tumor and endothelial cells . V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells .", "V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells . So far, there are no investigations on the role of endothelial v-ATPases for the process of tumor cell adhesion onto the endothelium. Thus, we were interested in the consequences of the inhibition of endothelial v-ATPase by archazolid on the interaction between endothelial and cancer cells. Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins.", "Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins. For the first time, our study revealed a link between the function of v-ATPases and the adhesion and transmigration properties of endothelial cells. CellTiter-Blue Cell Viability Assay Promega, Mannheim, Germany was performed according to the manufacturer's protocol for determining the cell viability of cells after treatment with archazolid. This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment.", "This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment. Fluorescence was measured with an Infinite F200 pro microplate reader Tecan, Männedorf, Switzerland at 560 nm excitation and 590 nm emission . CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega was performed according to the manufacturer's instructions for determining the lactate dehydrogenase LDH release after treatment with archazolid. Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells.", "Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells. Released LDH catalyzes the enzymatic conversion of lactate to pyruvate which provides NADH for the conversion of iodonitrotetrazolium violet into a red formazan product in the presence of diaphorase. The absorbance was measured with a Varioskan Flash microplate reader Thermo Fisher Scientific at 490 nm. LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes.", "LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes. Both dyes were incubated for 10 min at 37˚C before acquisition of single images by a Leica DMI6000 B fluorescence microscope Leica Microsystems, Wetzlar, Germany . HUVECs were seeded in collagen G-coated 24-well plates and grown to confluence for two days before treatment. The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ .", "The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ . Tumor cell adhesion was determined by fluorescence measurements with an Infinite F200 pro microplate reader Tecan at 485 nm excitation and 535 nm emission . For blocking the integrin β1 subunit on MDA-MB-231 or PC-3 cells, CellTracker Greenlabeled MDA-MB-231 or PC-3 cells were incubated with an anti-integrin β1 antibody P5D2, ab24693, Abcam, Cambridge, United Kingdom at a concentration of 1 μg antibody per one million cells in 1 ml DMEM. Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM .", "Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM . HUVECs were washed once with ECGM before untreated MDA-MB-231 or PC-3 cells were added to HUVECs. For the adhesion of MDA-MB-231 or PC-3 cells onto extracellular matrix ECM components 24-well plates were coated with collagen G 10 μg/ml in PBS , human plasma fibronectin 10 μg/ml PBS or laminin-411 10 μg/ml in Dulbecco's PBS DPBS containing Ca 2+ and Mg 2+ at 4˚C overnight. The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated.", "The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated. Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye as described in the section cell adhesion assay and resuspended in medium 199 PAN-Biotech containing 0.1% BSA. HUVECs were washed twice with medium 199 containing 0.1% BSA before MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Medium 199 containing 0.1% BSA was used as negative control and medium 199 containing 20% FCS was used as chemoattractant for transmigration in the lower compartment. Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission .", "Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission . HUVECs were grown to confluence on 6-well plates before they were treated with archazolid for 12 h. The cells were induced to upregulate the gene expression of cell adhesion molecules by TNFα. RNA was isolated using the RNeasy Mini Kit from Qiagen Hilden, Germany according to the manufacturer's protocol. On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific .", "On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific . qPCR experiments were performed using a StepOnePlus System Applied Biosystems, Thermo Fisher Scientific and data was analyzed by the StepOne and Ste-pOnePlus Software v2.3. Power SYBR Green PCR Master Mix Life Technologies and the comparative C T quantitation method 2 -ΔΔCT were used. HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany .", "HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany . In the case of ICAM-1 the detached cells were fixed with 4% formaldehyde Polysciences, Hirschberg an der Bergstraße, Germany in PBS for 10 min and washed once with PBS before incubating with the fluorescein isothiocyanate FITC -labeled anti-human CD54 mouse, ICAM-1 antibody MCA1615F, Biozol, Eching, Germany at room temperature for 45 min. For all other proteins, the cells were not fixed and washed once with PBS before incubating with the antibodies phycoerythrin PE -labeled anti-human CD106 mouse, VCAM-1 , PE-labeled anti-human CD62E mouse, E-selectin and PE-labeled anti-human CD325 mouse, N-cadherin from Becton Dickinson on ice for 45 min. These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany .", "These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany . To stain the surface collagen on HUVECs, cells were incubated with an anti-human collagen antibody rabbit, 1:40, ab36064, Abcam on ice for 30 min. The staining procedure was performed on ice to ensure that surface proteins or antibodies are not endocytosed. The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei.", "The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei. Confluent HUVECs in 6-well plates were treated as indicated. Cells were washed with ice-cold PBS and lysed with RIPA buffer supplemented with protease inhibitors Complete Mini EDTA-free; Roche, Mannheim, Germany , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride, β-glycerophosphate, sodium pyrophosphate and H 2 O 2 . Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany .", "Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany . Separated proteins were transferred onto polyvinylidene difluoride membranes by tank blotting Bio-Rad Laboratories for immunodetection. Membranes were blocked with 5% boltinggrade milk powder Carl Roth in TBS containing 0.1% Tween 20 Sigma-Aldrich . The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis.", "The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis. Cathepsin B activity assay was performed as described in the publication by Kubisch et al. . Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride.", "Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride. The fluorogenic cathepsin B substrate Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride C5429, Sigma-Aldrich was added to 30 μg of the cell lysate diluted in assay buffer supplemented with 2 mM L-cysteine C7880, Sigma-Aldrich and incubated for 30 min at 40˚C. Fluorescence was measured at 348 nm excitation and 440 nm emission with a microplate reader Varioskan Flash, Thermo Fisher Scientific . The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction.", "The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction. The HUVEC Nucleofector Kit Lonza, Cologne, Germany was used to transfect HUVECs. The transfection was performed according to the manufacturer's protocol using 2.5 μg plasmid DNA for 500,000 cells Nucleofector 2b Device, Lonza . 48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe .", "48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe . hCathepsin B was digested with PmeI and XbaI and the linear DNA fragment not corresponding to the human CTSB gene was religated to generate the empty pcDNA3.1 - delta MCS plasmid that was used for control transfections. The original backbone of hCathepsin B is the pcDNA3.1 - from Thermo Fisher Scientific. The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA .", "The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA . One-way ANOVA followed by Tukey's post-hoc test or unpaired t-test was used for the evaluation of a minimum of three independent experiments. The numbers of independently performed experiments n are stated in the corresponding figure legends. p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM .", "p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM . Since the v-ATPase inhibitor archazolid has never been used for studies in endothelial cells, we first performed cytotoxicity assays. We treated confluent HUVECs with up to 1 nM archazolid for 24 and 48 h and observed that this treatment has neither an influence on the metabolic activity nor on the release of LDH after 24 h Fig 1A and 1B, left panels . The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings.", "The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings. Microscopic analysis revealed that also the integrity of the endothelial monolayer was not affected by archazolid, but the cells showed a slightly different morphology Fig 2A : Archazolid-treated cells were swollen compared to control cells, which was not unexpected, as vacuolation of the endoplasmic reticulum ER has been described for other cell types and is typical for v-ATPase inhibitors . This effect was obvious both in subconfluent and in confluent cells Fig 2A . Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells.", "Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells. Thus, this dye can serve as an indicator of v-ATPase inhibition. To proof that archazolid is also functionally active as a v-ATPase inhibitor in HUVECs, cells were treated with 1 nM archazolid before they were incubated with LysoTracker Red DND-99 and Hoechst 33342. As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase.", "As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase. We analyzed the adhesion of MDA-MB-231 cells onto HUVECs. Confluent HUVECs were treated with up to 1 nM archazolid for 24 h. Untreated MDA-MB-231 cells were labeled with Cell-Tracker Green CMFDA Dye. Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells.", "Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells. HUVECs seeded in a Boyden chamber were treated with 1 nM archazolid for 24 h. CellTracker Green-labeled MDA-MB-231 cells not treated with archazolid were allowed to transmigrate through the endothelial monolayer for 24 h. As shown in Fig 3C, archazolid significantly decreased the transendothelial migration of MDA-MB-231 cells. The influence of archazolid on tumor cell adhesion was not only studied in HUVECs, which represent macrovascular endothelial cells, but also in microvascular HMEC-1 cells. Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B .", "Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B . Of note, the adhesion of non-metastatic Jurkat cells, an acute T cell leukemia cell line, remained unaffected after treatment of HUVECs with archazolid S1A Fig . Taken together, archazolid treatment augmented the adhesive properties of both micro-and macrovascular endothelial cells for metastatic tumor cells, but not for non-metastatic ones. Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process.", "Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process. Ligands for the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin were found to be expressed on tumor cells and to mediate tumor-endothelial cell interaction . Inhibition of the v-ATPase might affect the expression of endothelial cell adhesion molecules on mRNA or protein levels. To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A .", "To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A . The protein expression of these adhesion molecules on the surface of endothelial cells was analyzed by flow cytometry. Archazolid 1 nM, 24 h did not affect the cell surface expression of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5B . Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners.", "Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners. Within this protein family β1-integrins have been reported to mediate tumor cell adhesion onto quiescent endothelial cells . In order to elucidate the role of β1-integrins for the archazolid-induced tumor cell adhesion, the integrin β1-subunit was blocked either on MDA-MB-231 cells, PC-3 cells or on HUVECs. Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels .", "Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels . Depending on their α subunit, β1-integrins have a variety of ligands including extracellular matrix ECM components such as collagen, fibronectin and laminin . Therefore, we hypothesized that archazolid treatment of endothelial cells might lead to an upregulation of these components. MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A .", "MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A . MDA-MB-231 and PC-3 cells also adhered to fibronectin-coated plastic, but to a much lesser extent compared to the collagen coating. Therefore, we focused on the interaction between these two tumor cell lines and collagen. Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix.", "Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix. To detect collagen on the endothelial surface, archazolid-treated HUVECs were labeled with an antibody against collagen type I-IV on ice to prevent endocytosis and to ensure that the antibody does not bind to intracellular collagen. Interestingly, archazolid increased the amount of surface collagen on HUVECs by about 50% Fig 7C . Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen .", "Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen . As collagen is degraded by cathepsin B and the activation of cathepsin B depends on v-ATPase activity 28, 42 , we suggested that an accumulation of collagen on the surface of endothelial cells might be a consequence of an impaired functionality of cathepsin B. Therefore, an enzyme activity assay based on the proteolysis of a fluorogenic cathepsin B substrate was performed. In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A .", "In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A . In line with this result, western blot analysis showed that archazolid 1 nM reduces the protein expression of the mature, active form of cathepsin B to less than 40% of the control in HUVECs Fig 8B . To proof whether the archazolid-induced tumor cell adhesion is a consequence of the decreased amount of cathepsin B, HUVECs were transfected with a plasmid coding for human cathepsin B or with the empty vector as control. After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A .", "After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A . Overexpression of cathepsin B strongly diminished both the basal and the archazolid-induced adhesion of MDA-MB-231 cells Fig 9B . Targeting the proton pump v-ATPase for cancer therapy has gained great interest since its inhibition was reported to reduce the invasiveness of cancer cells and, most importantly, also metastasis . Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions.", "Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions. For the first time, we were able to show a link between v-ATPase and the adhesion and transmigration properties of the endothelium. Inhibition of the v-ATPase in endothelial cells by archazolid significantly increased the adhesion of metastatic cancer cells and decreased the transendothelial migration of cancer cells which was attributed to augmented collagen levels on the surface on archazolid-treated endothelial cells. Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound.", "Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound. However, we found that the archazolid-induced tumor cell adhesion was mediated by β1-integrins expressed on MDA-MB-231 breast cancer and PC-3 prostate cancer cells as blocking of the integrin β1 subunit on these tumor cells reversed the pro-adhesive effect of archazolid. In adhesion experiments on plastic coated with extracellular matrix components, we could show that MDA-MB-231 and PC-3 cells clearly favored the interaction with collagen, whereas the adhesion of non-metastatic Jurkat cells was largely independent from extracellular matrix proteins S1B Fig . The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins .", "The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins . α4β1integrins are receptors for VCAM-1 and fibronectin and it has been shown that Jurkat cells interact with human endothelial cells that express VCAM-1 after cytokine treatment or cells transfected with VCAM-1 . Our results are in line with previous studies showing that α2β1-and α3β1-integrin expressing MDA-MB-231 and PC-3 cells were able to rapidly attach to collagen in the cortical bone matrix. In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin .", "In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin . Though expressing receptors for fibronectin and laminin, MDA-MB-231 and PC-3 cells adhered to fibronectin to a much lesser extent and did not adhere to laminin, probably due to lower affinities to these extracellular matrix components. Importantly, v-ATPase inhibition by archazolid increased the surface levels of the extracellular matrix component collagen, which might explain that the increase of MDA-MB-231 and PC-3 cells onto archazolid-treated HUVECs is independent of endothelial cell adhesion molecules. By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation .", "By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation . In addition, recent studies reported that v-ATPase inhibition impairs the activity of cathepsin B in cancer cells . Therefore, we suggested that the accumulation of collagen on the endothelial surface might be a consequence of impaired cathepsin B activity or expression in endothelial cells. In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes.", "In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes. Following transport to the Golgi apparatus, the preprocathepsin B is glycosylated with mannose-containing oligosaccharides. The targeting of procathepsin B to lysosomes is mannose-6-phosphate receptor-dependent and its dissociation from the receptor as well as its proteolytic processing into mature cathepsin B requires acidification of the compartment . In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism.", "In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism. Interestingly, overexpression of cathepsin B attenuated the archazolid-induced adhesion of breast cancer cells onto endothelial cells, indicating that the adhesion negatively correlates with the expression of cathepsin B. As cathepsin B can also degrade other extracellular matrix components such as fibronectin and laminin , v-ATPase inhibition could lead to an accumulation of these proteins and an increased adhesion of cells expressing fibronectin or laminin receptors. However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig .", "However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig . Interestingly . In hepatic cancer cells, archazolid reduces Ras/Raf/MEK/ERK signaling by altering the membrane composition and fluidity . We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells.", "We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells. As a result, the amount of collagen on the surface of endothelial cells was significantly upregulated, which finally resulted in an increased adhesion of the β1-integrin-expressing metastatic cancer cell lines MDA-MB-231 and PC-3 onto archazolidtreated endothelial cells, whereas the adhesion of non-metastatic Jurkat cells was unaffected. This study shows that the v-ATPase plays an important role in regulating the adhesion of cells expressing receptors for extracellular matrix components. Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells.", "Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells. The fact that the adhesion of metastatic tumor cells onto endothelial cells is increased while their transendothelial migration is reduced upon inhibition of endothelial v-ATPase by archazolid further supports the view of archazolid as a potential anti-metastatic compound." ]

[ "The vacuolar-type H + -ATPase v-ATPase is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells ECs , in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process.", "The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.", "Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound. Text: The vacuolar-type H + -ATPase v-ATPase is the major proton pump responsible for acidification of intracellular compartments in eukaryotic cells . The enzyme consists of two multi-subunit complexes, the soluble V 1 transmembrane V o subcomplex required for the proton transport across membranes . In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing.", "In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing. In specialized cell types including osteoclasts and renal epithelial cells, v-ATPases can also be expressed on the plasma membrane, where they pump protons into the extracellular space . In cancer cells v-ATPases are expressed on the plasma membrane in order to eliminate toxic cytosolic H + . Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years.", "Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years. Archazolid A and B are highly potent and specific inhibitors of v-ATPases . They were first isolated from the myxobacterium Archangium gephyra . These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development.", "These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development. Archazolids can be either produced by fermentation or by total synthesis . In the field of cancer research several studies reported on interesting pharmacological effects of archazolid: It reduced the migration of different invasive tumor cells in vitro and cancer cell metastasis in vivo in a breast tumor mouse model . Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression .", "Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression . Up to now, the role of v-ATPases in endothelial cells has only rarely been investigated. The endothelium plays a crucial role in the pathogenesis and progression of cancer: The metastatic cascade includes local angiogenesis at the site of the primary tumor and adhesion of tumor cells at the site of metastasis . Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth .", "Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth . Circulating cancer cells can adhere to the endothelium at distant sites. This adhesive interaction is mediated by receptors and corresponding ligands expressed on tumor and endothelial cells . V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells .", "V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells . So far, there are no investigations on the role of endothelial v-ATPases for the process of tumor cell adhesion onto the endothelium. Thus, we were interested in the consequences of the inhibition of endothelial v-ATPase by archazolid on the interaction between endothelial and cancer cells. Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins.", "Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins. For the first time, our study revealed a link between the function of v-ATPases and the adhesion and transmigration properties of endothelial cells. CellTiter-Blue Cell Viability Assay Promega, Mannheim, Germany was performed according to the manufacturer's protocol for determining the cell viability of cells after treatment with archazolid. This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment.", "This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment. Fluorescence was measured with an Infinite F200 pro microplate reader Tecan, Männedorf, Switzerland at 560 nm excitation and 590 nm emission . CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega was performed according to the manufacturer's instructions for determining the lactate dehydrogenase LDH release after treatment with archazolid. Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells.", "Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells. Released LDH catalyzes the enzymatic conversion of lactate to pyruvate which provides NADH for the conversion of iodonitrotetrazolium violet into a red formazan product in the presence of diaphorase. The absorbance was measured with a Varioskan Flash microplate reader Thermo Fisher Scientific at 490 nm. LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes.", "LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes. Both dyes were incubated for 10 min at 37˚C before acquisition of single images by a Leica DMI6000 B fluorescence microscope Leica Microsystems, Wetzlar, Germany . HUVECs were seeded in collagen G-coated 24-well plates and grown to confluence for two days before treatment. The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ .", "The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ . Tumor cell adhesion was determined by fluorescence measurements with an Infinite F200 pro microplate reader Tecan at 485 nm excitation and 535 nm emission . For blocking the integrin β1 subunit on MDA-MB-231 or PC-3 cells, CellTracker Greenlabeled MDA-MB-231 or PC-3 cells were incubated with an anti-integrin β1 antibody P5D2, ab24693, Abcam, Cambridge, United Kingdom at a concentration of 1 μg antibody per one million cells in 1 ml DMEM. Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM .", "Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM . HUVECs were washed once with ECGM before untreated MDA-MB-231 or PC-3 cells were added to HUVECs. For the adhesion of MDA-MB-231 or PC-3 cells onto extracellular matrix ECM components 24-well plates were coated with collagen G 10 μg/ml in PBS , human plasma fibronectin 10 μg/ml PBS or laminin-411 10 μg/ml in Dulbecco's PBS DPBS containing Ca 2+ and Mg 2+ at 4˚C overnight. The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated.", "The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated. Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye as described in the section cell adhesion assay and resuspended in medium 199 PAN-Biotech containing 0.1% BSA. HUVECs were washed twice with medium 199 containing 0.1% BSA before MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Medium 199 containing 0.1% BSA was used as negative control and medium 199 containing 20% FCS was used as chemoattractant for transmigration in the lower compartment. Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission .", "Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission . HUVECs were grown to confluence on 6-well plates before they were treated with archazolid for 12 h. The cells were induced to upregulate the gene expression of cell adhesion molecules by TNFα. RNA was isolated using the RNeasy Mini Kit from Qiagen Hilden, Germany according to the manufacturer's protocol. On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific .", "On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific . qPCR experiments were performed using a StepOnePlus System Applied Biosystems, Thermo Fisher Scientific and data was analyzed by the StepOne and Ste-pOnePlus Software v2.3. Power SYBR Green PCR Master Mix Life Technologies and the comparative C T quantitation method 2 -ΔΔCT were used. HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany .", "HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany . In the case of ICAM-1 the detached cells were fixed with 4% formaldehyde Polysciences, Hirschberg an der Bergstraße, Germany in PBS for 10 min and washed once with PBS before incubating with the fluorescein isothiocyanate FITC -labeled anti-human CD54 mouse, ICAM-1 antibody MCA1615F, Biozol, Eching, Germany at room temperature for 45 min. For all other proteins, the cells were not fixed and washed once with PBS before incubating with the antibodies phycoerythrin PE -labeled anti-human CD106 mouse, VCAM-1 , PE-labeled anti-human CD62E mouse, E-selectin and PE-labeled anti-human CD325 mouse, N-cadherin from Becton Dickinson on ice for 45 min. These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany .", "These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany . To stain the surface collagen on HUVECs, cells were incubated with an anti-human collagen antibody rabbit, 1:40, ab36064, Abcam on ice for 30 min. The staining procedure was performed on ice to ensure that surface proteins or antibodies are not endocytosed. The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei.", "The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei. Confluent HUVECs in 6-well plates were treated as indicated. Cells were washed with ice-cold PBS and lysed with RIPA buffer supplemented with protease inhibitors Complete Mini EDTA-free; Roche, Mannheim, Germany , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride, β-glycerophosphate, sodium pyrophosphate and H 2 O 2 . Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany .", "Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany . Separated proteins were transferred onto polyvinylidene difluoride membranes by tank blotting Bio-Rad Laboratories for immunodetection. Membranes were blocked with 5% boltinggrade milk powder Carl Roth in TBS containing 0.1% Tween 20 Sigma-Aldrich . The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis.", "The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis. Cathepsin B activity assay was performed as described in the publication by Kubisch et al. . Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride.", "Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride. The fluorogenic cathepsin B substrate Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride C5429, Sigma-Aldrich was added to 30 μg of the cell lysate diluted in assay buffer supplemented with 2 mM L-cysteine C7880, Sigma-Aldrich and incubated for 30 min at 40˚C. Fluorescence was measured at 348 nm excitation and 440 nm emission with a microplate reader Varioskan Flash, Thermo Fisher Scientific . The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction.", "The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction. The HUVEC Nucleofector Kit Lonza, Cologne, Germany was used to transfect HUVECs. The transfection was performed according to the manufacturer's protocol using 2.5 μg plasmid DNA for 500,000 cells Nucleofector 2b Device, Lonza . 48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe .", "48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe . hCathepsin B was digested with PmeI and XbaI and the linear DNA fragment not corresponding to the human CTSB gene was religated to generate the empty pcDNA3.1 - delta MCS plasmid that was used for control transfections. The original backbone of hCathepsin B is the pcDNA3.1 - from Thermo Fisher Scientific. The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA .", "The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA . One-way ANOVA followed by Tukey's post-hoc test or unpaired t-test was used for the evaluation of a minimum of three independent experiments. The numbers of independently performed experiments n are stated in the corresponding figure legends. p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM .", "p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM . Since the v-ATPase inhibitor archazolid has never been used for studies in endothelial cells, we first performed cytotoxicity assays. We treated confluent HUVECs with up to 1 nM archazolid for 24 and 48 h and observed that this treatment has neither an influence on the metabolic activity nor on the release of LDH after 24 h Fig 1A and 1B, left panels . The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings.", "The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings. Microscopic analysis revealed that also the integrity of the endothelial monolayer was not affected by archazolid, but the cells showed a slightly different morphology Fig 2A : Archazolid-treated cells were swollen compared to control cells, which was not unexpected, as vacuolation of the endoplasmic reticulum ER has been described for other cell types and is typical for v-ATPase inhibitors . This effect was obvious both in subconfluent and in confluent cells Fig 2A . Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells.", "Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells. Thus, this dye can serve as an indicator of v-ATPase inhibition. To proof that archazolid is also functionally active as a v-ATPase inhibitor in HUVECs, cells were treated with 1 nM archazolid before they were incubated with LysoTracker Red DND-99 and Hoechst 33342. As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase.", "As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase. We analyzed the adhesion of MDA-MB-231 cells onto HUVECs. Confluent HUVECs were treated with up to 1 nM archazolid for 24 h. Untreated MDA-MB-231 cells were labeled with Cell-Tracker Green CMFDA Dye. Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells.", "Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells. HUVECs seeded in a Boyden chamber were treated with 1 nM archazolid for 24 h. CellTracker Green-labeled MDA-MB-231 cells not treated with archazolid were allowed to transmigrate through the endothelial monolayer for 24 h. As shown in Fig 3C, archazolid significantly decreased the transendothelial migration of MDA-MB-231 cells. The influence of archazolid on tumor cell adhesion was not only studied in HUVECs, which represent macrovascular endothelial cells, but also in microvascular HMEC-1 cells. Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B .", "Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B . Of note, the adhesion of non-metastatic Jurkat cells, an acute T cell leukemia cell line, remained unaffected after treatment of HUVECs with archazolid S1A Fig . Taken together, archazolid treatment augmented the adhesive properties of both micro-and macrovascular endothelial cells for metastatic tumor cells, but not for non-metastatic ones. Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process.", "Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process. Ligands for the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin were found to be expressed on tumor cells and to mediate tumor-endothelial cell interaction . Inhibition of the v-ATPase might affect the expression of endothelial cell adhesion molecules on mRNA or protein levels. To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A .", "To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A . The protein expression of these adhesion molecules on the surface of endothelial cells was analyzed by flow cytometry. Archazolid 1 nM, 24 h did not affect the cell surface expression of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5B . Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners.", "Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners. Within this protein family β1-integrins have been reported to mediate tumor cell adhesion onto quiescent endothelial cells . In order to elucidate the role of β1-integrins for the archazolid-induced tumor cell adhesion, the integrin β1-subunit was blocked either on MDA-MB-231 cells, PC-3 cells or on HUVECs. Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels .", "Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels . Depending on their α subunit, β1-integrins have a variety of ligands including extracellular matrix ECM components such as collagen, fibronectin and laminin . Therefore, we hypothesized that archazolid treatment of endothelial cells might lead to an upregulation of these components. MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A .", "MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A . MDA-MB-231 and PC-3 cells also adhered to fibronectin-coated plastic, but to a much lesser extent compared to the collagen coating. Therefore, we focused on the interaction between these two tumor cell lines and collagen. Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix.", "Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix. To detect collagen on the endothelial surface, archazolid-treated HUVECs were labeled with an antibody against collagen type I-IV on ice to prevent endocytosis and to ensure that the antibody does not bind to intracellular collagen. Interestingly, archazolid increased the amount of surface collagen on HUVECs by about 50% Fig 7C . Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen .", "Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen . As collagen is degraded by cathepsin B and the activation of cathepsin B depends on v-ATPase activity 28, 42 , we suggested that an accumulation of collagen on the surface of endothelial cells might be a consequence of an impaired functionality of cathepsin B. Therefore, an enzyme activity assay based on the proteolysis of a fluorogenic cathepsin B substrate was performed. In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A .", "In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A . In line with this result, western blot analysis showed that archazolid 1 nM reduces the protein expression of the mature, active form of cathepsin B to less than 40% of the control in HUVECs Fig 8B . To proof whether the archazolid-induced tumor cell adhesion is a consequence of the decreased amount of cathepsin B, HUVECs were transfected with a plasmid coding for human cathepsin B or with the empty vector as control. After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A .", "After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A . Overexpression of cathepsin B strongly diminished both the basal and the archazolid-induced adhesion of MDA-MB-231 cells Fig 9B . Targeting the proton pump v-ATPase for cancer therapy has gained great interest since its inhibition was reported to reduce the invasiveness of cancer cells and, most importantly, also metastasis . Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions.", "Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions. For the first time, we were able to show a link between v-ATPase and the adhesion and transmigration properties of the endothelium. Inhibition of the v-ATPase in endothelial cells by archazolid significantly increased the adhesion of metastatic cancer cells and decreased the transendothelial migration of cancer cells which was attributed to augmented collagen levels on the surface on archazolid-treated endothelial cells. Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound.", "Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound. However, we found that the archazolid-induced tumor cell adhesion was mediated by β1-integrins expressed on MDA-MB-231 breast cancer and PC-3 prostate cancer cells as blocking of the integrin β1 subunit on these tumor cells reversed the pro-adhesive effect of archazolid. In adhesion experiments on plastic coated with extracellular matrix components, we could show that MDA-MB-231 and PC-3 cells clearly favored the interaction with collagen, whereas the adhesion of non-metastatic Jurkat cells was largely independent from extracellular matrix proteins S1B Fig . The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins .", "The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins . α4β1integrins are receptors for VCAM-1 and fibronectin and it has been shown that Jurkat cells interact with human endothelial cells that express VCAM-1 after cytokine treatment or cells transfected with VCAM-1 . Our results are in line with previous studies showing that α2β1-and α3β1-integrin expressing MDA-MB-231 and PC-3 cells were able to rapidly attach to collagen in the cortical bone matrix. In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin .", "In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin . Though expressing receptors for fibronectin and laminin, MDA-MB-231 and PC-3 cells adhered to fibronectin to a much lesser extent and did not adhere to laminin, probably due to lower affinities to these extracellular matrix components. Importantly, v-ATPase inhibition by archazolid increased the surface levels of the extracellular matrix component collagen, which might explain that the increase of MDA-MB-231 and PC-3 cells onto archazolid-treated HUVECs is independent of endothelial cell adhesion molecules. By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation .", "By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation . In addition, recent studies reported that v-ATPase inhibition impairs the activity of cathepsin B in cancer cells . Therefore, we suggested that the accumulation of collagen on the endothelial surface might be a consequence of impaired cathepsin B activity or expression in endothelial cells. In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes.", "In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes. Following transport to the Golgi apparatus, the preprocathepsin B is glycosylated with mannose-containing oligosaccharides. The targeting of procathepsin B to lysosomes is mannose-6-phosphate receptor-dependent and its dissociation from the receptor as well as its proteolytic processing into mature cathepsin B requires acidification of the compartment . In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism.", "In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism. Interestingly, overexpression of cathepsin B attenuated the archazolid-induced adhesion of breast cancer cells onto endothelial cells, indicating that the adhesion negatively correlates with the expression of cathepsin B. As cathepsin B can also degrade other extracellular matrix components such as fibronectin and laminin , v-ATPase inhibition could lead to an accumulation of these proteins and an increased adhesion of cells expressing fibronectin or laminin receptors. However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig .", "However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig . Interestingly . In hepatic cancer cells, archazolid reduces Ras/Raf/MEK/ERK signaling by altering the membrane composition and fluidity . We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells.", "We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells. As a result, the amount of collagen on the surface of endothelial cells was significantly upregulated, which finally resulted in an increased adhesion of the β1-integrin-expressing metastatic cancer cell lines MDA-MB-231 and PC-3 onto archazolidtreated endothelial cells, whereas the adhesion of non-metastatic Jurkat cells was unaffected. This study shows that the v-ATPase plays an important role in regulating the adhesion of cells expressing receptors for extracellular matrix components. Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells.", "Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells. The fact that the adhesion of metastatic tumor cells onto endothelial cells is increased while their transendothelial migration is reduced upon inhibition of endothelial v-ATPase by archazolid further supports the view of archazolid as a potential anti-metastatic compound." ]

[ "The vacuolar-type H + -ATPase v-ATPase is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells ECs , in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process.", "The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.", "Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound. Text: The vacuolar-type H + -ATPase v-ATPase is the major proton pump responsible for acidification of intracellular compartments in eukaryotic cells . The enzyme consists of two multi-subunit complexes, the soluble V 1 transmembrane V o subcomplex required for the proton transport across membranes . In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing.", "In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing. In specialized cell types including osteoclasts and renal epithelial cells, v-ATPases can also be expressed on the plasma membrane, where they pump protons into the extracellular space . In cancer cells v-ATPases are expressed on the plasma membrane in order to eliminate toxic cytosolic H + . Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years.", "Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years. Archazolid A and B are highly potent and specific inhibitors of v-ATPases . They were first isolated from the myxobacterium Archangium gephyra . These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development.", "These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development. Archazolids can be either produced by fermentation or by total synthesis . In the field of cancer research several studies reported on interesting pharmacological effects of archazolid: It reduced the migration of different invasive tumor cells in vitro and cancer cell metastasis in vivo in a breast tumor mouse model . Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression .", "Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression . Up to now, the role of v-ATPases in endothelial cells has only rarely been investigated. The endothelium plays a crucial role in the pathogenesis and progression of cancer: The metastatic cascade includes local angiogenesis at the site of the primary tumor and adhesion of tumor cells at the site of metastasis . Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth .", "Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth . Circulating cancer cells can adhere to the endothelium at distant sites. This adhesive interaction is mediated by receptors and corresponding ligands expressed on tumor and endothelial cells . V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells .", "V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells . So far, there are no investigations on the role of endothelial v-ATPases for the process of tumor cell adhesion onto the endothelium. Thus, we were interested in the consequences of the inhibition of endothelial v-ATPase by archazolid on the interaction between endothelial and cancer cells. Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins.", "Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins. For the first time, our study revealed a link between the function of v-ATPases and the adhesion and transmigration properties of endothelial cells. CellTiter-Blue Cell Viability Assay Promega, Mannheim, Germany was performed according to the manufacturer's protocol for determining the cell viability of cells after treatment with archazolid. This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment.", "This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment. Fluorescence was measured with an Infinite F200 pro microplate reader Tecan, Männedorf, Switzerland at 560 nm excitation and 590 nm emission . CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega was performed according to the manufacturer's instructions for determining the lactate dehydrogenase LDH release after treatment with archazolid. Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells.", "Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells. Released LDH catalyzes the enzymatic conversion of lactate to pyruvate which provides NADH for the conversion of iodonitrotetrazolium violet into a red formazan product in the presence of diaphorase. The absorbance was measured with a Varioskan Flash microplate reader Thermo Fisher Scientific at 490 nm. LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes.", "LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes. Both dyes were incubated for 10 min at 37˚C before acquisition of single images by a Leica DMI6000 B fluorescence microscope Leica Microsystems, Wetzlar, Germany . HUVECs were seeded in collagen G-coated 24-well plates and grown to confluence for two days before treatment. The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ .", "The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ . Tumor cell adhesion was determined by fluorescence measurements with an Infinite F200 pro microplate reader Tecan at 485 nm excitation and 535 nm emission . For blocking the integrin β1 subunit on MDA-MB-231 or PC-3 cells, CellTracker Greenlabeled MDA-MB-231 or PC-3 cells were incubated with an anti-integrin β1 antibody P5D2, ab24693, Abcam, Cambridge, United Kingdom at a concentration of 1 μg antibody per one million cells in 1 ml DMEM. Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM .", "Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM . HUVECs were washed once with ECGM before untreated MDA-MB-231 or PC-3 cells were added to HUVECs. For the adhesion of MDA-MB-231 or PC-3 cells onto extracellular matrix ECM components 24-well plates were coated with collagen G 10 μg/ml in PBS , human plasma fibronectin 10 μg/ml PBS or laminin-411 10 μg/ml in Dulbecco's PBS DPBS containing Ca 2+ and Mg 2+ at 4˚C overnight. The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated.", "The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated. Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye as described in the section cell adhesion assay and resuspended in medium 199 PAN-Biotech containing 0.1% BSA. HUVECs were washed twice with medium 199 containing 0.1% BSA before MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Medium 199 containing 0.1% BSA was used as negative control and medium 199 containing 20% FCS was used as chemoattractant for transmigration in the lower compartment. Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission .", "Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission . HUVECs were grown to confluence on 6-well plates before they were treated with archazolid for 12 h. The cells were induced to upregulate the gene expression of cell adhesion molecules by TNFα. RNA was isolated using the RNeasy Mini Kit from Qiagen Hilden, Germany according to the manufacturer's protocol. On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific .", "On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific . qPCR experiments were performed using a StepOnePlus System Applied Biosystems, Thermo Fisher Scientific and data was analyzed by the StepOne and Ste-pOnePlus Software v2.3. Power SYBR Green PCR Master Mix Life Technologies and the comparative C T quantitation method 2 -ΔΔCT were used. HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany .", "HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany . In the case of ICAM-1 the detached cells were fixed with 4% formaldehyde Polysciences, Hirschberg an der Bergstraße, Germany in PBS for 10 min and washed once with PBS before incubating with the fluorescein isothiocyanate FITC -labeled anti-human CD54 mouse, ICAM-1 antibody MCA1615F, Biozol, Eching, Germany at room temperature for 45 min. For all other proteins, the cells were not fixed and washed once with PBS before incubating with the antibodies phycoerythrin PE -labeled anti-human CD106 mouse, VCAM-1 , PE-labeled anti-human CD62E mouse, E-selectin and PE-labeled anti-human CD325 mouse, N-cadherin from Becton Dickinson on ice for 45 min. These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany .", "These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany . To stain the surface collagen on HUVECs, cells were incubated with an anti-human collagen antibody rabbit, 1:40, ab36064, Abcam on ice for 30 min. The staining procedure was performed on ice to ensure that surface proteins or antibodies are not endocytosed. The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei.", "The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei. Confluent HUVECs in 6-well plates were treated as indicated. Cells were washed with ice-cold PBS and lysed with RIPA buffer supplemented with protease inhibitors Complete Mini EDTA-free; Roche, Mannheim, Germany , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride, β-glycerophosphate, sodium pyrophosphate and H 2 O 2 . Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany .", "Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany . Separated proteins were transferred onto polyvinylidene difluoride membranes by tank blotting Bio-Rad Laboratories for immunodetection. Membranes were blocked with 5% boltinggrade milk powder Carl Roth in TBS containing 0.1% Tween 20 Sigma-Aldrich . The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis.", "The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis. Cathepsin B activity assay was performed as described in the publication by Kubisch et al. . Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride.", "Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride. The fluorogenic cathepsin B substrate Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride C5429, Sigma-Aldrich was added to 30 μg of the cell lysate diluted in assay buffer supplemented with 2 mM L-cysteine C7880, Sigma-Aldrich and incubated for 30 min at 40˚C. Fluorescence was measured at 348 nm excitation and 440 nm emission with a microplate reader Varioskan Flash, Thermo Fisher Scientific . The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction.", "The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction. The HUVEC Nucleofector Kit Lonza, Cologne, Germany was used to transfect HUVECs. The transfection was performed according to the manufacturer's protocol using 2.5 μg plasmid DNA for 500,000 cells Nucleofector 2b Device, Lonza . 48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe .", "48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe . hCathepsin B was digested with PmeI and XbaI and the linear DNA fragment not corresponding to the human CTSB gene was religated to generate the empty pcDNA3.1 - delta MCS plasmid that was used for control transfections. The original backbone of hCathepsin B is the pcDNA3.1 - from Thermo Fisher Scientific. The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA .", "The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA . One-way ANOVA followed by Tukey's post-hoc test or unpaired t-test was used for the evaluation of a minimum of three independent experiments. The numbers of independently performed experiments n are stated in the corresponding figure legends. p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM .", "p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM . Since the v-ATPase inhibitor archazolid has never been used for studies in endothelial cells, we first performed cytotoxicity assays. We treated confluent HUVECs with up to 1 nM archazolid for 24 and 48 h and observed that this treatment has neither an influence on the metabolic activity nor on the release of LDH after 24 h Fig 1A and 1B, left panels . The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings.", "The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings. Microscopic analysis revealed that also the integrity of the endothelial monolayer was not affected by archazolid, but the cells showed a slightly different morphology Fig 2A : Archazolid-treated cells were swollen compared to control cells, which was not unexpected, as vacuolation of the endoplasmic reticulum ER has been described for other cell types and is typical for v-ATPase inhibitors . This effect was obvious both in subconfluent and in confluent cells Fig 2A . Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells.", "Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells. Thus, this dye can serve as an indicator of v-ATPase inhibition. To proof that archazolid is also functionally active as a v-ATPase inhibitor in HUVECs, cells were treated with 1 nM archazolid before they were incubated with LysoTracker Red DND-99 and Hoechst 33342. As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase.", "As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase. We analyzed the adhesion of MDA-MB-231 cells onto HUVECs. Confluent HUVECs were treated with up to 1 nM archazolid for 24 h. Untreated MDA-MB-231 cells were labeled with Cell-Tracker Green CMFDA Dye. Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells.", "Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells. HUVECs seeded in a Boyden chamber were treated with 1 nM archazolid for 24 h. CellTracker Green-labeled MDA-MB-231 cells not treated with archazolid were allowed to transmigrate through the endothelial monolayer for 24 h. As shown in Fig 3C, archazolid significantly decreased the transendothelial migration of MDA-MB-231 cells. The influence of archazolid on tumor cell adhesion was not only studied in HUVECs, which represent macrovascular endothelial cells, but also in microvascular HMEC-1 cells. Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B .", "Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B . Of note, the adhesion of non-metastatic Jurkat cells, an acute T cell leukemia cell line, remained unaffected after treatment of HUVECs with archazolid S1A Fig . Taken together, archazolid treatment augmented the adhesive properties of both micro-and macrovascular endothelial cells for metastatic tumor cells, but not for non-metastatic ones. Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process.", "Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process. Ligands for the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin were found to be expressed on tumor cells and to mediate tumor-endothelial cell interaction . Inhibition of the v-ATPase might affect the expression of endothelial cell adhesion molecules on mRNA or protein levels. To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A .", "To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A . The protein expression of these adhesion molecules on the surface of endothelial cells was analyzed by flow cytometry. Archazolid 1 nM, 24 h did not affect the cell surface expression of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5B . Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners.", "Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners. Within this protein family β1-integrins have been reported to mediate tumor cell adhesion onto quiescent endothelial cells . In order to elucidate the role of β1-integrins for the archazolid-induced tumor cell adhesion, the integrin β1-subunit was blocked either on MDA-MB-231 cells, PC-3 cells or on HUVECs. Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels .", "Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels . Depending on their α subunit, β1-integrins have a variety of ligands including extracellular matrix ECM components such as collagen, fibronectin and laminin . Therefore, we hypothesized that archazolid treatment of endothelial cells might lead to an upregulation of these components. MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A .", "MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A . MDA-MB-231 and PC-3 cells also adhered to fibronectin-coated plastic, but to a much lesser extent compared to the collagen coating. Therefore, we focused on the interaction between these two tumor cell lines and collagen. Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix.", "Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix. To detect collagen on the endothelial surface, archazolid-treated HUVECs were labeled with an antibody against collagen type I-IV on ice to prevent endocytosis and to ensure that the antibody does not bind to intracellular collagen. Interestingly, archazolid increased the amount of surface collagen on HUVECs by about 50% Fig 7C . Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen .", "Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen . As collagen is degraded by cathepsin B and the activation of cathepsin B depends on v-ATPase activity 28, 42 , we suggested that an accumulation of collagen on the surface of endothelial cells might be a consequence of an impaired functionality of cathepsin B. Therefore, an enzyme activity assay based on the proteolysis of a fluorogenic cathepsin B substrate was performed. In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A .", "In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A . In line with this result, western blot analysis showed that archazolid 1 nM reduces the protein expression of the mature, active form of cathepsin B to less than 40% of the control in HUVECs Fig 8B . To proof whether the archazolid-induced tumor cell adhesion is a consequence of the decreased amount of cathepsin B, HUVECs were transfected with a plasmid coding for human cathepsin B or with the empty vector as control. After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A .", "After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A . Overexpression of cathepsin B strongly diminished both the basal and the archazolid-induced adhesion of MDA-MB-231 cells Fig 9B . Targeting the proton pump v-ATPase for cancer therapy has gained great interest since its inhibition was reported to reduce the invasiveness of cancer cells and, most importantly, also metastasis . Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions.", "Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions. For the first time, we were able to show a link between v-ATPase and the adhesion and transmigration properties of the endothelium. Inhibition of the v-ATPase in endothelial cells by archazolid significantly increased the adhesion of metastatic cancer cells and decreased the transendothelial migration of cancer cells which was attributed to augmented collagen levels on the surface on archazolid-treated endothelial cells. Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound.", "Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound. However, we found that the archazolid-induced tumor cell adhesion was mediated by β1-integrins expressed on MDA-MB-231 breast cancer and PC-3 prostate cancer cells as blocking of the integrin β1 subunit on these tumor cells reversed the pro-adhesive effect of archazolid. In adhesion experiments on plastic coated with extracellular matrix components, we could show that MDA-MB-231 and PC-3 cells clearly favored the interaction with collagen, whereas the adhesion of non-metastatic Jurkat cells was largely independent from extracellular matrix proteins S1B Fig . The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins .", "The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins . α4β1integrins are receptors for VCAM-1 and fibronectin and it has been shown that Jurkat cells interact with human endothelial cells that express VCAM-1 after cytokine treatment or cells transfected with VCAM-1 . Our results are in line with previous studies showing that α2β1-and α3β1-integrin expressing MDA-MB-231 and PC-3 cells were able to rapidly attach to collagen in the cortical bone matrix. In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin .", "In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin . Though expressing receptors for fibronectin and laminin, MDA-MB-231 and PC-3 cells adhered to fibronectin to a much lesser extent and did not adhere to laminin, probably due to lower affinities to these extracellular matrix components. Importantly, v-ATPase inhibition by archazolid increased the surface levels of the extracellular matrix component collagen, which might explain that the increase of MDA-MB-231 and PC-3 cells onto archazolid-treated HUVECs is independent of endothelial cell adhesion molecules. By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation .", "By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation . In addition, recent studies reported that v-ATPase inhibition impairs the activity of cathepsin B in cancer cells . Therefore, we suggested that the accumulation of collagen on the endothelial surface might be a consequence of impaired cathepsin B activity or expression in endothelial cells. In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes.", "In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes. Following transport to the Golgi apparatus, the preprocathepsin B is glycosylated with mannose-containing oligosaccharides. The targeting of procathepsin B to lysosomes is mannose-6-phosphate receptor-dependent and its dissociation from the receptor as well as its proteolytic processing into mature cathepsin B requires acidification of the compartment . In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism.", "In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism. Interestingly, overexpression of cathepsin B attenuated the archazolid-induced adhesion of breast cancer cells onto endothelial cells, indicating that the adhesion negatively correlates with the expression of cathepsin B. As cathepsin B can also degrade other extracellular matrix components such as fibronectin and laminin , v-ATPase inhibition could lead to an accumulation of these proteins and an increased adhesion of cells expressing fibronectin or laminin receptors. However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig .", "However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig . Interestingly . In hepatic cancer cells, archazolid reduces Ras/Raf/MEK/ERK signaling by altering the membrane composition and fluidity . We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells.", "We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells. As a result, the amount of collagen on the surface of endothelial cells was significantly upregulated, which finally resulted in an increased adhesion of the β1-integrin-expressing metastatic cancer cell lines MDA-MB-231 and PC-3 onto archazolidtreated endothelial cells, whereas the adhesion of non-metastatic Jurkat cells was unaffected. This study shows that the v-ATPase plays an important role in regulating the adhesion of cells expressing receptors for extracellular matrix components. Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells.", "Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells. The fact that the adhesion of metastatic tumor cells onto endothelial cells is increased while their transendothelial migration is reduced upon inhibition of endothelial v-ATPase by archazolid further supports the view of archazolid as a potential anti-metastatic compound." ]

[ "The vacuolar-type H + -ATPase v-ATPase is the major proton pump that acidifies intracellular compartments of eukaryotic cells. Since the inhibition of v-ATPase resulted in anti-tumor and anti-metastatic effects in different tumor models, this enzyme has emerged as promising strategy against cancer. Here, we used the well-established v-ATPase inhibitor archazolid, a natural product first isolated from the myxobacterium Archangium gephyra, to study the consequences of v-ATPase inhibition in endothelial cells ECs , in particular on the interaction between ECs and cancer cells, which has been neglected so far. Human endothelial cells treated with archazolid showed an increased adhesion of tumor cells, whereas the transendothelial migration of tumor cells was reduced. The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process.", "The adhesion process was independent from the EC adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin. Instead, the adhesion was mediated by β1-integrins expressed on tumor cells, as blocking of the integrin β1 subunit reversed this process. Tumor cells preferentially adhered to the β1-integrin ligand collagen and archazolid led to an increase in the amount of collagen on the surface of ECs. The accumulation of collagen was accompanied by a strong decrease of the expression and activity of the protease cathepsin B. Overexpression of cathepsin B in ECs prevented the capability of archazolid to increase the adhesion of tumor cells onto ECs. Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound.", "Our study demonstrates that the inhibition of v-ATPase by archazolid induces a pro-adhesive phenotype in endothelial cells that promotes their interaction with cancer cells, whereas the transmigration of tumor cells was reduced. These findings further support archazolid as a promising anti-metastatic compound. Text: The vacuolar-type H + -ATPase v-ATPase is the major proton pump responsible for acidification of intracellular compartments in eukaryotic cells . The enzyme consists of two multi-subunit complexes, the soluble V 1 transmembrane V o subcomplex required for the proton transport across membranes . In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing.", "In most cell types v-ATPases are only expressed in the endomembrane system to regulate and maintain the acidic pH of intracellular compartments such as lysosomes, endosomes, the Golgi apparatus, secretory granules and coated vesicles . The function of v-ATPases is essential for cellular processes such as vesicular trafficking, receptor-mediated endocytosis and protein degradation and processing. In specialized cell types including osteoclasts and renal epithelial cells, v-ATPases can also be expressed on the plasma membrane, where they pump protons into the extracellular space . In cancer cells v-ATPases are expressed on the plasma membrane in order to eliminate toxic cytosolic H + . Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years.", "Most importantly, v-ATPases contribute to the acidic tumor microenvironment, which leads to the activation of proteases, thus facilitating tumor cell migration, invasion and angiogenesis . Since the inhibition of v-ATPase was shown to reduce the invasiveness of cancer cells and metastasis formation , this enzyme has emerged as a promising drug target in the recent years. Archazolid A and B are highly potent and specific inhibitors of v-ATPases . They were first isolated from the myxobacterium Archangium gephyra . These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development.", "These compounds inhibit v-ATPase at low nanomolar concentrations by binding to the subunit c of the V o complex. As their biological activity is comparable to the v-ATPase inhibitors bafilomycin and concanamycin , archazolids are natural compounds of high interest that can be used both as a tool to study the consequences of v-ATPase inhibition and as a lead for drug development. Archazolids can be either produced by fermentation or by total synthesis . In the field of cancer research several studies reported on interesting pharmacological effects of archazolid: It reduced the migration of different invasive tumor cells in vitro and cancer cell metastasis in vivo in a breast tumor mouse model . Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression .", "Furthermore, archazolid activated pathways of cellular stress response and apoptosis in highly invasive tumor cells . In classically activated macrophages, archazolid selectively induced the generation of tumor necrosis factor α TNFα , which may indirectly promote tumor suppression . Up to now, the role of v-ATPases in endothelial cells has only rarely been investigated. The endothelium plays a crucial role in the pathogenesis and progression of cancer: The metastatic cascade includes local angiogenesis at the site of the primary tumor and adhesion of tumor cells at the site of metastasis . Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth .", "Angiogenesis, the development of new blood vessels out of existing ones, depends on the proliferation, migration and differentiation of endothelial cells . This process ensures the nutrient supply of the tumor and its growth . Circulating cancer cells can adhere to the endothelium at distant sites. This adhesive interaction is mediated by receptors and corresponding ligands expressed on tumor and endothelial cells . V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells .", "V-ATPases have been reported to regulate intracellular pH and cell migration in microvascular endothelial cells . A recent study showed that the inhibition of v-ATPase by concanamycin prevented proliferation, reduced migration and impaired angiogenesis-related signaling in endothelial cells . So far, there are no investigations on the role of endothelial v-ATPases for the process of tumor cell adhesion onto the endothelium. Thus, we were interested in the consequences of the inhibition of endothelial v-ATPase by archazolid on the interaction between endothelial and cancer cells. Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins.", "Various cell adhesion molecules on the endothelium, such as intercellular adhesion molecule 1 ICAM-1 , vascular cell adhesion protein VCAM-1 , E-selectin or N-cadherin as well as integrins expressed on cancer cells have been reported to mediate cell adhesion of cancer cells onto endothelial cells . Accordingly, we focused on these cell adhesion molecules and integrins. For the first time, our study revealed a link between the function of v-ATPases and the adhesion and transmigration properties of endothelial cells. CellTiter-Blue Cell Viability Assay Promega, Mannheim, Germany was performed according to the manufacturer's protocol for determining the cell viability of cells after treatment with archazolid. This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment.", "This assay is based on the ability of metabolically active cells to reduce resazurin which results in fluorescent resorufin. The CellTiter-Blue Reagent was added to the cells 4 h before the endpoint of treatment. Fluorescence was measured with an Infinite F200 pro microplate reader Tecan, Männedorf, Switzerland at 560 nm excitation and 590 nm emission . CytoTox 96 Non-Radioactive Cytotoxicity Assay Promega was performed according to the manufacturer's instructions for determining the lactate dehydrogenase LDH release after treatment with archazolid. Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells.", "Lysis buffer was added to untreated cells 45 min before the end of treatment to induce the release of this enzyme. LDH is a cytosolic enzyme that is released by leaky cells. Released LDH catalyzes the enzymatic conversion of lactate to pyruvate which provides NADH for the conversion of iodonitrotetrazolium violet into a red formazan product in the presence of diaphorase. The absorbance was measured with a Varioskan Flash microplate reader Thermo Fisher Scientific at 490 nm. LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes.", "LysoTracker Red DND-99 Life Technologies, Thermo Fisher Scientific is a dye to measure pH values in viable cells. HUVECs were cultured to confluence on collagen G-coated μ-slides 80826, ibidi, Martinsried, Germany before they were treated with archazolid for 24 h. 1 μg/ ml Hoechst 33342 Sigma-Aldrich, Munich, Germany was used to visualize the nuclei and 50 nM LysoTracker Red DND-99 was used to visualize the acidic compartments which correspond to the lysosomes. Both dyes were incubated for 10 min at 37˚C before acquisition of single images by a Leica DMI6000 B fluorescence microscope Leica Microsystems, Wetzlar, Germany . HUVECs were seeded in collagen G-coated 24-well plates and grown to confluence for two days before treatment. The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ .", "The cells were incubated with indicated concentrations of archazolid for 24 h. Untreated MDA-MB-231 or PC-3 cells were labeled with CellTracker Green CMFDA Dye 5 μM in serum-free DMEM, 37˚C for 30 min before 100,000 cells per well were added to HUVECs and were allowed to adhere for various time points at 37˚C. Non-adherent tumor cells were washed off three times with PBS containing Ca 2+ and Mg 2+ . Tumor cell adhesion was determined by fluorescence measurements with an Infinite F200 pro microplate reader Tecan at 485 nm excitation and 535 nm emission . For blocking the integrin β1 subunit on MDA-MB-231 or PC-3 cells, CellTracker Greenlabeled MDA-MB-231 or PC-3 cells were incubated with an anti-integrin β1 antibody P5D2, ab24693, Abcam, Cambridge, United Kingdom at a concentration of 1 μg antibody per one million cells in 1 ml DMEM. Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM .", "Before adding to archazolid-treated HUVECs, MDA-MB-231 or PC-3 cells were washed once with DMEM. For blocking the integrin β1 subunit on HUVECs, the cells were incubated with the anti-integrin β1 antibody 0.1 μg/well in ECGM . HUVECs were washed once with ECGM before untreated MDA-MB-231 or PC-3 cells were added to HUVECs. For the adhesion of MDA-MB-231 or PC-3 cells onto extracellular matrix ECM components 24-well plates were coated with collagen G 10 μg/ml in PBS , human plasma fibronectin 10 μg/ml PBS or laminin-411 10 μg/ml in Dulbecco's PBS DPBS containing Ca 2+ and Mg 2+ at 4˚C overnight. The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated.", "The adhesion of MDA-MB-231 and PC-3 cells onto these three most prominent ECM components was carried out as described above 10 min adhesion at 37˚C . HUVECs were grown on a porous filter membrane Transwell insert, polycarbonate membrane, 8 μm pores; Corning, New York, USA for 48 h and were treated as indicated. Untreated MDA-MB-231 cells were labeled with CellTracker Green CMFDA Dye as described in the section cell adhesion assay and resuspended in medium 199 PAN-Biotech containing 0.1% BSA. HUVECs were washed twice with medium 199 containing 0.1% BSA before MDA-MB-231 cells were allowed to transmigrate through the endothelial monolayer for 24 h. Medium 199 containing 0.1% BSA was used as negative control and medium 199 containing 20% FCS was used as chemoattractant for transmigration in the lower compartment. Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission .", "Non-migrated cells remaining in the upper compartment were carefully removed using a cotton swab. Transmigrated cells were lysed in radioimmunoprecipitation assay RIPA buffer and transmigration was quantified by measuring the fluorescence signal at 485 nm excitation and 535 nm emission . HUVECs were grown to confluence on 6-well plates before they were treated with archazolid for 12 h. The cells were induced to upregulate the gene expression of cell adhesion molecules by TNFα. RNA was isolated using the RNeasy Mini Kit from Qiagen Hilden, Germany according to the manufacturer's protocol. On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific .", "On-column DNase digestion was performed to remove genomic DNA. RNA was transcribed into cDNA by Superscript II Life Technologies, Thermo Fisher Scientific . qPCR experiments were performed using a StepOnePlus System Applied Biosystems, Thermo Fisher Scientific and data was analyzed by the StepOne and Ste-pOnePlus Software v2.3. Power SYBR Green PCR Master Mix Life Technologies and the comparative C T quantitation method 2 -ΔΔCT were used. HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany .", "HUVECs were grown to confluence on 12-well plates before they were treated with archazolid for 24 h. Cells were treated with TNFα for 24 h to induce the expression of cell adhesion molecules. Subsequently, the cells were detached with HyClone HyQTase GE Healthcare, Freiburg, Germany . In the case of ICAM-1 the detached cells were fixed with 4% formaldehyde Polysciences, Hirschberg an der Bergstraße, Germany in PBS for 10 min and washed once with PBS before incubating with the fluorescein isothiocyanate FITC -labeled anti-human CD54 mouse, ICAM-1 antibody MCA1615F, Biozol, Eching, Germany at room temperature for 45 min. For all other proteins, the cells were not fixed and washed once with PBS before incubating with the antibodies phycoerythrin PE -labeled anti-human CD106 mouse, VCAM-1 , PE-labeled anti-human CD62E mouse, E-selectin and PE-labeled anti-human CD325 mouse, N-cadherin from Becton Dickinson on ice for 45 min. These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany .", "These antibodies were diluted in PBS containing 0.2% BSA. The surface expression of cell adhesion molecules was measured by flow cytometry FACSVerse, Becton Dickinson, Heidelberg, Germany . To stain the surface collagen on HUVECs, cells were incubated with an anti-human collagen antibody rabbit, 1:40, ab36064, Abcam on ice for 30 min. The staining procedure was performed on ice to ensure that surface proteins or antibodies are not endocytosed. The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei.", "The cells were washed once with PBS containing Ca 2+ and Mg 2+ before they were fixed with Roti-Histofix Carl Roth . Alexa Fluor 488-conjugated anti-rabbit antibody goat, 1:400, A11008, Life Technologies was used as secondary antibody and Hoechst 33342 1 μg/ml, Sigma-Aldrich was used to visualize nuclei. Confluent HUVECs in 6-well plates were treated as indicated. Cells were washed with ice-cold PBS and lysed with RIPA buffer supplemented with protease inhibitors Complete Mini EDTA-free; Roche, Mannheim, Germany , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride, β-glycerophosphate, sodium pyrophosphate and H 2 O 2 . Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany .", "Protein determination was performed using the Pierce BCA Protein Assay Kit Thermo Fisher Scientific . Equal amounts of proteins 10-20 μg were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis SDS-PAGE; Bio-Rad Laboratories, Munich, Germany . Separated proteins were transferred onto polyvinylidene difluoride membranes by tank blotting Bio-Rad Laboratories for immunodetection. Membranes were blocked with 5% boltinggrade milk powder Carl Roth in TBS containing 0.1% Tween 20 Sigma-Aldrich . The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis.", "The following antibodies were used: mouse anti-human cathepsin B antibody IM27L, Merck 1:500 , mouse anti-β-actin-peroxidase antibody A3854, Sigma-Aldrich 1:100,000 and antimouse IgG horse radish peroxidase HRP -linked antibody 7076, Cell Signaling, Frankfurt, Germany 1:5,000 . ImageJ version 1.49m was used for densitometric analysis. Cathepsin B activity assay was performed as described in the publication by Kubisch et al. . Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride.", "Confluent HUVECs or HMEC-1 seeded in 6-well plates were treated as indicated. Cells were washed with PBS and lysed with the non-denaturating M-PER mammalian protein extraction reagent 78501, Thermo Fisher Scientific supplemented with protease inhibitors Complete Mini EDTA-free, Roche , sodium orthovanadate, sodium fluoride, phenylmethylsulphonyl fluoride. The fluorogenic cathepsin B substrate Z-Arg-Arg-7-amido-4-methylcoumarin hydrochloride C5429, Sigma-Aldrich was added to 30 μg of the cell lysate diluted in assay buffer supplemented with 2 mM L-cysteine C7880, Sigma-Aldrich and incubated for 30 min at 40˚C. Fluorescence was measured at 348 nm excitation and 440 nm emission with a microplate reader Varioskan Flash, Thermo Fisher Scientific . The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction.", "The intensity of the fluorescence signal corresponded to the cathepsin B enzyme activity. For background subtraction the cathepsin B inhibitor CA-074Me Enzo Life Sciences, Lörrach, Germany was added to an additional reaction. The HUVEC Nucleofector Kit Lonza, Cologne, Germany was used to transfect HUVECs. The transfection was performed according to the manufacturer's protocol using 2.5 μg plasmid DNA for 500,000 cells Nucleofector 2b Device, Lonza . 48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe .", "48 h after transfection the cells were treated for further experiments. The addgene plasmid #11249 hCathepsin B was kindly provided by Hyeryun Choe . hCathepsin B was digested with PmeI and XbaI and the linear DNA fragment not corresponding to the human CTSB gene was religated to generate the empty pcDNA3.1 - delta MCS plasmid that was used for control transfections. The original backbone of hCathepsin B is the pcDNA3.1 - from Thermo Fisher Scientific. The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA .", "The control vector pcDNA3.1 - delta MCS used for our transfections was cloned on the basis of hCathepsin B and is therefore lacking almost the whole part of the multiple cloning site of the pcDNA3.1 - . Statistical analyses were performed using GraphPad Prism 5.0 San Diego, USA . One-way ANOVA followed by Tukey's post-hoc test or unpaired t-test was used for the evaluation of a minimum of three independent experiments. The numbers of independently performed experiments n are stated in the corresponding figure legends. p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM .", "p 0.05 was considered as statistically significant. Data are expressed as mean ± standard error of the mean SEM . Since the v-ATPase inhibitor archazolid has never been used for studies in endothelial cells, we first performed cytotoxicity assays. We treated confluent HUVECs with up to 1 nM archazolid for 24 and 48 h and observed that this treatment has neither an influence on the metabolic activity nor on the release of LDH after 24 h Fig 1A and 1B, left panels . The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings.", "The metabolic activity and the release of LDH were only slightly affected by the highest concentration of archazolid after 48 h Fig 1A and 1B, right panels . Consequently, the following experiments were all carried out after 24 h or less of archazolid treatment in order to exclude any cytotoxic effects of archazolid within our experimental settings. Microscopic analysis revealed that also the integrity of the endothelial monolayer was not affected by archazolid, but the cells showed a slightly different morphology Fig 2A : Archazolid-treated cells were swollen compared to control cells, which was not unexpected, as vacuolation of the endoplasmic reticulum ER has been described for other cell types and is typical for v-ATPase inhibitors . This effect was obvious both in subconfluent and in confluent cells Fig 2A . Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells.", "Inhibition of v-ATPase prevents the acidification of lysosomes . Using the cell-permeable dye LysoTracker Red DND-99, it is possible to label the acidic lysosomes in living cells. Thus, this dye can serve as an indicator of v-ATPase inhibition. To proof that archazolid is also functionally active as a v-ATPase inhibitor in HUVECs, cells were treated with 1 nM archazolid before they were incubated with LysoTracker Red DND-99 and Hoechst 33342. As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase.", "As shown in Fig 2B, the red vesicular staining corresponding to acidified lysosomes in control cells disappeared completely after treatment with archazolid. In summary, archazolid treatment for 24 h was not cytotoxic to quiescent HUVECs, but inhibited the functionality of the v-ATPase. We analyzed the adhesion of MDA-MB-231 cells onto HUVECs. Confluent HUVECs were treated with up to 1 nM archazolid for 24 h. Untreated MDA-MB-231 cells were labeled with Cell-Tracker Green CMFDA Dye. Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells.", "Interestingly, v-ATPase inhibition strongly increased the attachment of the metastatic breast carcinoma cell line MDA-MB-231 onto HUVECs after 10 and 120 min of adhesion Fig 3A and 3B . We also investigated the influence of archazolid on the transendothelial migration of MDA-MB-231 cells. HUVECs seeded in a Boyden chamber were treated with 1 nM archazolid for 24 h. CellTracker Green-labeled MDA-MB-231 cells not treated with archazolid were allowed to transmigrate through the endothelial monolayer for 24 h. As shown in Fig 3C, archazolid significantly decreased the transendothelial migration of MDA-MB-231 cells. The influence of archazolid on tumor cell adhesion was not only studied in HUVECs, which represent macrovascular endothelial cells, but also in microvascular HMEC-1 cells. Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B .", "Moreover, besides the breast cancer cell line MDA-MB-231, also PC-3 prostate cancer cells were used as a second metastatic cancer cell line. Archazolid treatment of endothelial cells increased the attachment of MDA-MB-231 cells onto the HMEC-1 monolayer after 120 min of adhesion Fig 4A and increased the attachment of PC-3 cells onto the HUVEC monolayer after 30 and 60 min of adhesion Fig 4B . Of note, the adhesion of non-metastatic Jurkat cells, an acute T cell leukemia cell line, remained unaffected after treatment of HUVECs with archazolid S1A Fig . Taken together, archazolid treatment augmented the adhesive properties of both micro-and macrovascular endothelial cells for metastatic tumor cells, but not for non-metastatic ones. Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process.", "Of note, cancer cell adhesion onto archazolid-activated endothelial cells increased with the time of adhesion. The adhesion of tumor cells onto the endothelium is in principle similar to that of leukocytes, but slightly differs in the molecules that mediate the adhesion process. Ligands for the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin were found to be expressed on tumor cells and to mediate tumor-endothelial cell interaction . Inhibition of the v-ATPase might affect the expression of endothelial cell adhesion molecules on mRNA or protein levels. To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A .", "To determine the mRNA expression of ICAM-1, VCAM-1, E-selectin and Ncadherin, HUVECs were treated with archazolid for 12 h. TNFα is known to upregulate the expression of ICAM-1, VCAM-1 and E-selectin and, thus, served as positive control. Quantitative real-time PCR showed that v-ATPase inhibition in HUVECs did not alter the mRNA levels of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5A . The protein expression of these adhesion molecules on the surface of endothelial cells was analyzed by flow cytometry. Archazolid 1 nM, 24 h did not affect the cell surface expression of ICAM-1, VCAM-1, E-selectin and N-cadherin Fig 5B . Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners.", "Besides ICAM-1, VCAM-1, E-selectin and N-cadherin, also integrins are able to mediate the process of cell adhesion . Since none of the cell adhesion molecules expressed on HUVECs were regulated upon archazolid treatment, we considered integrins as potential interaction partners. Within this protein family β1-integrins have been reported to mediate tumor cell adhesion onto quiescent endothelial cells . In order to elucidate the role of β1-integrins for the archazolid-induced tumor cell adhesion, the integrin β1-subunit was blocked either on MDA-MB-231 cells, PC-3 cells or on HUVECs. Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels .", "Of note, as in all experiments throughout this study, only endothelial cells were treated with archazolid. After blocking β1-integrins on MDA-MB-231 or PC-3 cells, the archazolid-induced tumor cell adhesion was reduced almost to control level Fig 6A and 6B , left panels , whereas blocking of β1-integrins on HUVECs had no significant effect on the increase of tumor cell adhesion by v-ATPase inhibition Fig 6A and 6B , right panels . Depending on their α subunit, β1-integrins have a variety of ligands including extracellular matrix ECM components such as collagen, fibronectin and laminin . Therefore, we hypothesized that archazolid treatment of endothelial cells might lead to an upregulation of these components. MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A .", "MDA-MB-231 and PC-3 cells were allowed to adhere onto plastic that was coated with these ECM components. This cell adhesion assay revealed that MDA-MB-231 as well as PC-3 cells favor the interaction with the ECM component collagen, as the adhesion onto collagen is much higher than onto the uncoated plastic control Fig 7A . MDA-MB-231 and PC-3 cells also adhered to fibronectin-coated plastic, but to a much lesser extent compared to the collagen coating. Therefore, we focused on the interaction between these two tumor cell lines and collagen. Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix.", "Blocking of the integrin β1 subunit on MDA-MB-231 and PC-3 cells clearly abolished the interaction with collagen Fig 7B , indicating that the attachment of these tumor cells to collagen is mediated by β1-integrins. Since collagen is the major ECM component MDA-MB-231 and PC-3 cells interact with, the next step was to prove whether v-ATPase inhibition influences the amount of collagen expressed by HUVECs as extracellular matrix. To detect collagen on the endothelial surface, archazolid-treated HUVECs were labeled with an antibody against collagen type I-IV on ice to prevent endocytosis and to ensure that the antibody does not bind to intracellular collagen. Interestingly, archazolid increased the amount of surface collagen on HUVECs by about 50% Fig 7C . Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen .", "Control stainings were performed using an antibody against the cytosolic p65 subunit of the transcription factor nuclear factor κB NFκB to show that intracellular proteins were not detected by this staining method S2 Fig . It was reported that v-ATPase inhibition by archazolid impairs the activity of cathepsin B , a lysosomal enzyme that degrades extracellular matrix components including collagen . As collagen is degraded by cathepsin B and the activation of cathepsin B depends on v-ATPase activity 28, 42 , we suggested that an accumulation of collagen on the surface of endothelial cells might be a consequence of an impaired functionality of cathepsin B. Therefore, an enzyme activity assay based on the proteolysis of a fluorogenic cathepsin B substrate was performed. In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A .", "In archazolid-treated HUVECs and HMEC-1 the activity of cathepsin B was induce both the mRNA 1 ng/ml TNF and the cell surface expression 10 ng/ml TNF of ICAM-1, VCAM-1, E-selectin and Ncadherin. Inhibition of endothelial vATPase increases tumor cell adhesion to endothelial cells strongly decreased by approximately 50% compared to control cells at an archazolid concentration of 1 nM Fig 8A . In line with this result, western blot analysis showed that archazolid 1 nM reduces the protein expression of the mature, active form of cathepsin B to less than 40% of the control in HUVECs Fig 8B . To proof whether the archazolid-induced tumor cell adhesion is a consequence of the decreased amount of cathepsin B, HUVECs were transfected with a plasmid coding for human cathepsin B or with the empty vector as control. After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A .", "After 48 h, the transfected cells were treated with 1 nM archazolid. The level of cathepsin B after transfection and treatment was assessed by western blot analysis Fig 9A . Overexpression of cathepsin B strongly diminished both the basal and the archazolid-induced adhesion of MDA-MB-231 cells Fig 9B . Targeting the proton pump v-ATPase for cancer therapy has gained great interest since its inhibition was reported to reduce the invasiveness of cancer cells and, most importantly, also metastasis . Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions.", "Thus, intensive research related to v-ATPases was done in cancer cells, whereas there are only few studies investigating v-ATPases in endothelial cells indicating a role in migration, proliferation and possibly angiogenesis . In the present study we used the myxobacterial natural product archazolid to investigate the consequences of v-ATPase inhibition in the endothelium on tumor-endothelial cell interactions. For the first time, we were able to show a link between v-ATPase and the adhesion and transmigration properties of the endothelium. Inhibition of the v-ATPase in endothelial cells by archazolid significantly increased the adhesion of metastatic cancer cells and decreased the transendothelial migration of cancer cells which was attributed to augmented collagen levels on the surface on archazolid-treated endothelial cells. Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound.", "Of note, adhesion of the non-metastatic Jurkat cell line onto archazolid-treated endothelial cells remained unaffected. The archazolidinduced adhesion of tumor cells was independent from the endothelial cell adhesion molecules ICAM-1, VCAM-1, E-selectin and N-cadherin, as their expression was not regulated by the compound. However, we found that the archazolid-induced tumor cell adhesion was mediated by β1-integrins expressed on MDA-MB-231 breast cancer and PC-3 prostate cancer cells as blocking of the integrin β1 subunit on these tumor cells reversed the pro-adhesive effect of archazolid. In adhesion experiments on plastic coated with extracellular matrix components, we could show that MDA-MB-231 and PC-3 cells clearly favored the interaction with collagen, whereas the adhesion of non-metastatic Jurkat cells was largely independent from extracellular matrix proteins S1B Fig . The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins .", "The different adhesion properties of metastatic cancer cells and Jurkat cells might be a result of the distinct integrin expression pattern of each cell line. MDA-MB-231 and PC-3 cells express α2β1-and α3β1-integrins, which represent collagen receptors , while Jurkat cells express α4β1-integrins but lack α2β1-, α3β1-integrins . α4β1integrins are receptors for VCAM-1 and fibronectin and it has been shown that Jurkat cells interact with human endothelial cells that express VCAM-1 after cytokine treatment or cells transfected with VCAM-1 . Our results are in line with previous studies showing that α2β1-and α3β1-integrin expressing MDA-MB-231 and PC-3 cells were able to rapidly attach to collagen in the cortical bone matrix. In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin .", "In contrast, Jurkat cells were not able to adhere and might preferentially interact with cell adhesion molecules rather than with ECM proteins. α2β1-and α3β1-integrins can additionally act as laminin receptors and at least α3β1integrins recognize fibronectin . Though expressing receptors for fibronectin and laminin, MDA-MB-231 and PC-3 cells adhered to fibronectin to a much lesser extent and did not adhere to laminin, probably due to lower affinities to these extracellular matrix components. Importantly, v-ATPase inhibition by archazolid increased the surface levels of the extracellular matrix component collagen, which might explain that the increase of MDA-MB-231 and PC-3 cells onto archazolid-treated HUVECs is independent of endothelial cell adhesion molecules. By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation .", "By performing a live cell proteolysis assay, Cavallo-Medved et al. demonstrated ECM degradation, in particular of gelatin and collagen IV, in association with active cathepsin B in caveolae of endothelial cells during tube formation . In addition, recent studies reported that v-ATPase inhibition impairs the activity of cathepsin B in cancer cells . Therefore, we suggested that the accumulation of collagen on the endothelial surface might be a consequence of impaired cathepsin B activity or expression in endothelial cells. In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes.", "In fact, we confirmed the impairment of cathepsin B activity by archazolid as the expression levels of the mature active form of this enzyme was strongly reduced. Cathepsin B is synthesized as preprocathepsin B on membrane-bound ribosomes. Following transport to the Golgi apparatus, the preprocathepsin B is glycosylated with mannose-containing oligosaccharides. The targeting of procathepsin B to lysosomes is mannose-6-phosphate receptor-dependent and its dissociation from the receptor as well as its proteolytic processing into mature cathepsin B requires acidification of the compartment . In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism.", "In cancer cells v-ATPase inhibition by archazolid impaired the mannose-6-phosphate receptor-mediated trafficking from the trans-Golgi network to prelysosomal compartments resulting in a decrease of active lysosomal proteases like cathepsin B . We assumed that the archazolid-induced decrease in cathepsin B activity and expression was based on the same mechanism. Interestingly, overexpression of cathepsin B attenuated the archazolid-induced adhesion of breast cancer cells onto endothelial cells, indicating that the adhesion negatively correlates with the expression of cathepsin B. As cathepsin B can also degrade other extracellular matrix components such as fibronectin and laminin , v-ATPase inhibition could lead to an accumulation of these proteins and an increased adhesion of cells expressing fibronectin or laminin receptors. However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig .", "However, we did not focus on these ECM components since they were not relevant for the adhesion of MDA-MB-231 and PC-3 cells. These cells predominantly adhered to collagen, while the adhesion of Jurkat cells is mostly independent from the ECM proteins collagen, fibronectin or laminin S1B Fig . Interestingly . In hepatic cancer cells, archazolid reduces Ras/Raf/MEK/ERK signaling by altering the membrane composition and fluidity . We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells.", "We assume that archazolid affects endothelial cells in a similar way leading to inhibition of Ras signaling and, therefore, reduced transendothelial migration of MDA-MB-231 cells. Taken together, our study shows that archazolid reduces the activity and expression of cathepsin B in endothelial cells. As a result, the amount of collagen on the surface of endothelial cells was significantly upregulated, which finally resulted in an increased adhesion of the β1-integrin-expressing metastatic cancer cell lines MDA-MB-231 and PC-3 onto archazolidtreated endothelial cells, whereas the adhesion of non-metastatic Jurkat cells was unaffected. This study shows that the v-ATPase plays an important role in regulating the adhesion of cells expressing receptors for extracellular matrix components. Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells.", "Archazolid represents a promising tool to elucidate the role of v-ATPase in endothelial cells. Moreover, we for the first time linked the function of v-ATPase to the adhesion and transmigration of tumor cells onto endothelial cells as well as to the remodeling of the extracellular matrix on the surface of endothelial cells. The fact that the adhesion of metastatic tumor cells onto endothelial cells is increased while their transendothelial migration is reduced upon inhibition of endothelial v-ATPase by archazolid further supports the view of archazolid as a potential anti-metastatic compound." ]

[ "BACKGROUND: Gastroenteritis GE and respiratory tract infection RTI outbreaks are a significant issue in nursing homes. This study aimed to describe GE and RTI outbreaks with infection and all-cause lethality rates according to the individual characteristics of nursing home residents. METHODS: Clinical and virological surveillance were conducted 2007 to 2018 . Virus stratifications for the analysis were: outbreaks with positive norovirus or influenza identifications respectively NoV+ or Flu+ , episodes with no NoV or influenza identification or testing respectively NoV- or Flu- . Associations between individual variables sex, age, length of stay LOS , autonomy status and infection and lethality rates were tested with univariate and Mantel-Haenszel MH methods. RESULTS: 61 GE outbreaks and 76 RTI oubreaks total 137 outbreaks were recorded involving respectively 4309 and 5862 residents.", "Associations between individual variables sex, age, length of stay LOS , autonomy status and infection and lethality rates were tested with univariate and Mantel-Haenszel MH methods. RESULTS: 61 GE outbreaks and 76 RTI oubreaks total 137 outbreaks were recorded involving respectively 4309 and 5862 residents. In univariate analysis, higher infection rates and age were associated in NoV+, NoV-, and Flu+ contexts, and lower infection rates were associated with longer stays NoV+ and NoV- . In MH stratified analysis virus, sex female/male adjusted for LOS <4 or ≥4 years , the odds of being infected remained significant among older residents ≥86 years : NoV+/male Odds ratio OR MH : 1.64, 95% confidence interval CI : 1.16–2.30 and Flu+/female and male respectively OR MH : 1.50, CI: 1.27–1.79 and 1.73, CI: 1.28–2.33 . In univariate analysis, lower autonomy status NoV+, Flu+ and Flu- and increased age Flu+ were associated with higher lethality. In MH adjusted analysis, significant OR age adjusted for autonomy was: Flu+/ ≥86 years compared with <86 years, 1.97 1.19–3.25 and OR autonomy adjusted for age for the more autonomous group compared with the less autonomous group was: Flu+, 0.41 0.24–0.69 ; Flu-, 0.42 0.20, 0.90 .", "In univariate analysis, lower autonomy status NoV+, Flu+ and Flu- and increased age Flu+ were associated with higher lethality. In MH adjusted analysis, significant OR age adjusted for autonomy was: Flu+/ ≥86 years compared with <86 years, 1.97 1.19–3.25 and OR autonomy adjusted for age for the more autonomous group compared with the less autonomous group was: Flu+, 0.41 0.24–0.69 ; Flu-, 0.42 0.20, 0.90 . CONCLUSION: The residents of nursing homes are increasingly elderly and dependent. The specific infection and lethality risks according to these two factors indicate that surveillance and infection control measures are essential and of high priority. Text: Introduction Gastroenteritis GE and respiratory tract infection RTI outbreaks represent a significant burden of illness in nursing homes. Viruses cause the majority of these outbreaks, and noroviruses and influenza viruses are the most common pathogens .", "Text: Introduction Gastroenteritis GE and respiratory tract infection RTI outbreaks represent a significant burden of illness in nursing homes. Viruses cause the majority of these outbreaks, and noroviruses and influenza viruses are the most common pathogens . Previous studies have suggested that viral respiratory infections and norovirus outbreaks are a common cause of hospitalization or death, particularly among elderly individuals . The impact of outbreaks has been described in terms of both frequency and epidemiology, but little is known about infection rates and all-cause lethality in GE and RTI nursing home outbreaks in relation to the individual characteristics of the residents . The residents of these institutions are increasingly elderly and dependent, and the impact of this trend on the seasonal outbreak burden requires in-depth investigation. The results of studies focused on this issue could yield valuable information for nursing homes, allowing them to adapt their infection control strategies, in particular for improved assessment of infection risk.", "The residents of these institutions are increasingly elderly and dependent, and the impact of this trend on the seasonal outbreak burden requires in-depth investigation. The results of studies focused on this issue could yield valuable information for nursing homes, allowing them to adapt their infection control strategies, in particular for improved assessment of infection risk. Our objective was to describe GE and RTI infection and all-cause lethality rates according to the individual characteristics of nursing home residents sex, age, length of stay, autonomy status , and to identify specific susceptibility patterns related to these types of viral outbreaks in these facilities. The present study explored outbreaks in 14 sites 28 units with geriatric nursing home activities for a total of 1121 beds caring for dependent people in southern Alsace an area in northeastern France . Data were collected between September 2007 and August 2018 . Each site was geographically independent and autonomous for social and care management.", "Data were collected between September 2007 and August 2018 . Each site was geographically independent and autonomous for social and care management. Units were located within the larger sites and were defined as a place having a dedicated team at one location. During outbreaks at one site, only the residents in the units with confirmed cases were included. Outbreak inclusion depended on institutional alert to the hygiene team. Surveillance was done in each unit independently, and the members of staff had to inform a physician or charge nurse when two or more potential related cases of pneumonia or GE were observed within four days and when three or more cases were observed for other RTI.", "Outbreak inclusion depended on institutional alert to the hygiene team. Surveillance was done in each unit independently, and the members of staff had to inform a physician or charge nurse when two or more potential related cases of pneumonia or GE were observed within four days and when three or more cases were observed for other RTI. Units also had to inform the hygiene team when these threshold values were exceeded. For influenza, the first suspected case led to a local alert and the hygiene team was contacted. A practitioner from the hygiene team collected the information and evaluated the clinical signs, the virology information and the epidemiological context with the physician in the affected unit. The detected cluster was only put under surveillance if the hygiene team considered that there was a potential outbreak phenomenon.", "A practitioner from the hygiene team collected the information and evaluated the clinical signs, the virology information and the epidemiological context with the physician in the affected unit. The detected cluster was only put under surveillance if the hygiene team considered that there was a potential outbreak phenomenon. The duration of 4 days was in relation with the national protocol with alert to the authorities when 5 cases occurred within 4 days . On a local level and in addition to the clusters reported to the authorities, clusters with at least 3 cases within a period of seven days in one unit could be recorded if they were reported to the hygiene team. Because several outbreaks could potentially occur in the same unit during the surveillance period, a resident could be included repeatedly in different clusters. As a result, the observed patterns reflected the characteristics of an institutional population with longitudinal and pluriannual exposures.", "Because several outbreaks could potentially occur in the same unit during the surveillance period, a resident could be included repeatedly in different clusters. As a result, the observed patterns reflected the characteristics of an institutional population with longitudinal and pluriannual exposures. Personal information and clinical information was collected by a practitioner from the hygiene team directly from the residents' health care records. Personal information was collected for all those present the first day of the outbreak. The collected information included: month and year of birth, sex, date of arrival at the nursing home and autonomy status. The autonomy status of residents in French nursing homes is assessed using the AGGIR scale Autonomy Gerontology Groups Iso-Resources , which is the legal instrument for evaluating dependency in the elderly and whose primary purpose is the allocation of means and resources .", "The collected information included: month and year of birth, sex, date of arrival at the nursing home and autonomy status. The autonomy status of residents in French nursing homes is assessed using the AGGIR scale Autonomy Gerontology Groups Iso-Resources , which is the legal instrument for evaluating dependency in the elderly and whose primary purpose is the allocation of means and resources . With the AGGIR scale, autonomy is classified into 6 Iso-Resource Groups GIR : GIR 1 bedridden or armchair-bound persons, mental functions seriously altered and requiring continuous presence , GIR 2 bedridden or armchair-bound persons, mental functions not totally altered and requiring assistance in most activities of daily living, or mental functions altered with preserved ability to get around , GIR 3 preserved mental autonomy with partially preserved motor autonomy and assistance several times a day for physical autonomy , GIR 4 moves around the home and sometimes assistance for washing, dressing, physical activities or eating , GIR 5 only occasional assistance for washing, meal preparation, and housework , GIR 6 autonomy for essential tasks of daily living . In outbreaks where the influenza virus was identified, influenza vaccination status and oseltamivir prescriptions were recorded as well. A file is transmitted in the Supporting Information with all previous data S1 Data . GE was defined as the sudden onset of vomiting and/or diarrhea over a 24 h period: i diarrhea �3 episodes, ii and/or vomiting �3 episodes, iii or diarrhea or vomiting <3 episodes with two or more other symptoms diarrhea, vomiting, stomach ache, abdominal cramps, nausea, fever, mucus in stools .", "A file is transmitted in the Supporting Information with all previous data S1 Data . GE was defined as the sudden onset of vomiting and/or diarrhea over a 24 h period: i diarrhea �3 episodes, ii and/or vomiting �3 episodes, iii or diarrhea or vomiting <3 episodes with two or more other symptoms diarrhea, vomiting, stomach ache, abdominal cramps, nausea, fever, mucus in stools . RTI presentation in older adults may be atypical, like for other acute illnesses in this age group . We used the recommended definitions for RTI surveillance in geriatric units, divided in 3 subcategories: i common cold syndromes or pharyngitis at least two of the following criteria: runny nose or sneezing, stuffy nose i.e. congestion , sore throat or hoarseness or difficulty swallowing, dry cough, swollen or tender glands in the neck cervical lymphadenopathy , ii influenza-like illness both the following criteria must be met: fever AND at least three other symptoms chills, new headache or eye pain, myalgia or body aches, malaise or loss of appetite, sore throat, new or increased dry cough and iii lower respiratory tract infection both of the following criteria must be met: at least two respiratory signs or symptoms new or increased cough, new/increased sputum production, O 2 saturation <94% or reduced >3% from baseline, abnormal lung examination new or changed , pleuritic chest pain, respiratory rate �25 breaths/min AND one or more constitutional signs/symptoms fever, leukocytosis, confusion, acute functional decline . Infection corresponding to one of these three subcategories was included in this study and classified as RTI.", "congestion , sore throat or hoarseness or difficulty swallowing, dry cough, swollen or tender glands in the neck cervical lymphadenopathy , ii influenza-like illness both the following criteria must be met: fever AND at least three other symptoms chills, new headache or eye pain, myalgia or body aches, malaise or loss of appetite, sore throat, new or increased dry cough and iii lower respiratory tract infection both of the following criteria must be met: at least two respiratory signs or symptoms new or increased cough, new/increased sputum production, O 2 saturation <94% or reduced >3% from baseline, abnormal lung examination new or changed , pleuritic chest pain, respiratory rate �25 breaths/min AND one or more constitutional signs/symptoms fever, leukocytosis, confusion, acute functional decline . Infection corresponding to one of these three subcategories was included in this study and classified as RTI. For both infection types, the practitioner from the hygiene team obtained clinical information from the patient's health care records, and members of the health care team were consulted if necessary to complete any missing information. At the end of the episode within seven days after the last identified case , case inclusion as exposed and not infected ENI or exposed and infected EI was determined with a resident physician. In order to study the lethality, the presence of each infected resident was evaluated once at least 56 days after the last case of each outbreak. Each resident was followed up retrospectively during eighth 7-day interval I n, n = 1 to 8, total 56 days, between the date of onset of symptoms and the fifty-sixth day of the studied period with three different possibilities: present alive and officially residing in the institution , lost to follow-up alive at the date of departure but no longer residing in the institution return home, transfer to another institution or death death recorded in the health care record .", "In order to study the lethality, the presence of each infected resident was evaluated once at least 56 days after the last case of each outbreak. Each resident was followed up retrospectively during eighth 7-day interval I n, n = 1 to 8, total 56 days, between the date of onset of symptoms and the fifty-sixth day of the studied period with three different possibilities: present alive and officially residing in the institution , lost to follow-up alive at the date of departure but no longer residing in the institution return home, transfer to another institution or death death recorded in the health care record . The dates of death and lost to follow-up were recorded. Testing for the virus was not systematic and was decided by the physicians in each institution in the presence of clinical signs. For GE, stool samples were sent to the National Reference Centre for Gastroenteritis Viruses in Dijon for laboratory testing, as previously described . For RTI surveillance, rapid tests were used to identify the influenza virus.", "For GE, stool samples were sent to the National Reference Centre for Gastroenteritis Viruses in Dijon for laboratory testing, as previously described . For RTI surveillance, rapid tests were used to identify the influenza virus. The rapid immunoassay diagnosis tests used for influenza detection were: Clearview1 Exact Influenza A and B Inverness Medical, Cologne, Germany from 2007 to 2014 and InfluenzaTop1 Alldiag, Strasbourg, France from 2014 to 2018. Given the low sensitivity of influenza rapid tests, they were no longer used once the control measures had been implemented and the influenza outbreak was under control. Samples were also occasionally sent to hospital laboratories or to the National Reference Centre for Influenza Viruses to detect viruses with real-time RT-PCR . Most testing targeted the norovirus NoV and influenza virus, but other tests were occasionally performed by the National Reference Centre for Influenza Viruses rhinovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza 1, 2, 3 and 4, and coronavirus and the National Reference Centre for Enteric Viruses rotavirus, astrovirus, and adenovirus .", "Samples were also occasionally sent to hospital laboratories or to the National Reference Centre for Influenza Viruses to detect viruses with real-time RT-PCR . Most testing targeted the norovirus NoV and influenza virus, but other tests were occasionally performed by the National Reference Centre for Influenza Viruses rhinovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza 1, 2, 3 and 4, and coronavirus and the National Reference Centre for Enteric Viruses rotavirus, astrovirus, and adenovirus . Because testing for the viruses was variable from one institution/physician to another, not used in some outbreaks, types of virus sought and considering the poor sensitivity of the rapid influenza tests, these two sources of data were used to define the epidemiological context of confirmed outbreaks. Consequently, individual cases were included consistently in all episodes according to clinical signs and medical evaluation. When virus testing was negative, the clinical signs were recorded and medical evaluation was used as previously to classify the included residents as infected or not infected. The epidemiological context of each outbreak was defined according to whether the virus had been identified or not.", "When virus testing was negative, the clinical signs were recorded and medical evaluation was used as previously to classify the included residents as infected or not infected. The epidemiological context of each outbreak was defined according to whether the virus had been identified or not. One or more positive samples led to the qualification of a NoV NoV+ or flu Flu+ context. The other episodes were qualified as flu or NoV outbreaks with no specific identification or testing NoV-and Flu- . Flu and NoV contexts did not eliminate other potential enteric or respiratory pathogens. Sex, age, length of stay LOS, in years and autonomy status were described for all exposed residents.", "Flu and NoV contexts did not eliminate other potential enteric or respiratory pathogens. Sex, age, length of stay LOS, in years and autonomy status were described for all exposed residents. Influenza vaccination and oseltamivir administration rates were calculated for confirmed influenza outbreaks. Dichotomous or categorical variables were expressed as percentages. In univariate analysis, the categories were specific in order to obtain a precise description of the age and LOS variables. Class intervals were 5 years for age and one year for LOS. The residents classified as GIR 4 to 6 sometimes, occasional and no assistance were grouped together because they were few.", "Class intervals were 5 years for age and one year for LOS. The residents classified as GIR 4 to 6 sometimes, occasional and no assistance were grouped together because they were few. For the multi-level analysis with 2x2 tables, a median value was used to define the two-level age categories. For LOS, assessing the longest stays was necessary to identify the effect of longer exposure in a nursing home. Consequently, a four-year cutoff was chosen to create the two categories. For autonomy, the two most dependent categories GIR � 2 were grouped together and compared with the more autonomous categories GIR � 3 .", "Consequently, a four-year cutoff was chosen to create the two categories. For autonomy, the two most dependent categories GIR � 2 were grouped together and compared with the more autonomous categories GIR � 3 . The outbreak epidemiological contexts were used with the four categories: NoV+, Flu+, NoV-and flu-. Other GE or RTI viruses were occasionally identified, but the number of results was too limited to develop separate analyses. However, all the results are available in the tables about the virus investigations along with NoV and influenza identifications. For the different categories, infection rate EI/Exposed Residents ER , in percentage was calculated according to sex, age group, LOS and autonomy status.", "However, all the results are available in the tables about the virus investigations along with NoV and influenza identifications. For the different categories, infection rate EI/Exposed Residents ER , in percentage was calculated according to sex, age group, LOS and autonomy status. To investigate all-cause lethality and define the appropriate period for the 56-day monitoring D 1 to 56 , the all-cause lethality rate per 7-day interval LR n /I n, n = 1 to 8 was calculated: number of deaths during interval I n / EI alive the first day of n th studied interval minus lost to follow-up EI during the interval I n � 100 . Seeing as successive clusters could occur within the same site, potentially influencing allcause lethality, the serial interval in days SI d was calculated. The SI d was the time period between the onset of symptoms of the last case in initial outbreak N and the onset of symptoms of the first case in the following outbreak N+1 . Investigations were performed when SI d was shorter or equal to the length of the previous D 1 to 56 and the following parameters were evaluated for these specific situations: number of episodes, residents infected in both outbreaks, and death among the identified individuals.", "The SI d was the time period between the onset of symptoms of the last case in initial outbreak N and the onset of symptoms of the first case in the following outbreak N+1 . Investigations were performed when SI d was shorter or equal to the length of the previous D 1 to 56 and the following parameters were evaluated for these specific situations: number of episodes, residents infected in both outbreaks, and death among the identified individuals. Finally, according to the death rate and the impact of successive outbreaks, the number of 7-day intervals N.I n to take into account was defined, and the all-cause lethality rate was analyzed during these periods I 1 to N th .I n or D 1 to 7 � N . All-cause lethality rates were calculated with the following formula: number of deaths from D 1 to 7 � N / EI number at D 1 minus lost to follow-up among EI during the period D 1 to 7 � N � 100 . Estimation of the turnover rate per 7-day interval among the infected residents was calculated on the base of the LOS median in years with the following formula: proportion of discharged residents: 50.0% in the case of the median / median LOS � 365 /7 . The average rate of residents discharged per 7-day period was calculated: number of lost to follow up during the period D 1 to 7 � N /number of exposed and infected residents at D 1 /N 7-day interval � 100.", "Estimation of the turnover rate per 7-day interval among the infected residents was calculated on the base of the LOS median in years with the following formula: proportion of discharged residents: 50.0% in the case of the median / median LOS � 365 /7 . The average rate of residents discharged per 7-day period was calculated: number of lost to follow up during the period D 1 to 7 � N /number of exposed and infected residents at D 1 /N 7-day interval � 100. As some residents were included in several outbreaks during the surveillance, the observations were not completely independent; non-parametric tests were used as a result. In univariate analysis, Chi-square or Fisher exact tests expected number of frequencies fewer than 5 were used to compare infection and lethality rates according to the studied parameters and the odds ratio was calculated by median-unbiased estimation. The Kruskal-Wallis test was used to compare median values. Confidence intervals for medians were calculated with bootstrap methods.", "The Kruskal-Wallis test was used to compare median values. Confidence intervals for medians were calculated with bootstrap methods. Covariate adjusted analyses were performed with two tables 2x2 . The respective impact of each individual factor was tested with Mantel-Haenszel chi-squared tests. The equality of the stratum odds ratios was tested with the Woolf test of homogeneity. Finally, for each virus context, multiple tables 2x2 were generated and tested with confounding variables, effect modifiers or covariables. Statistical analyses were done using R for Mas OS X version R 3.4.1 software with RStudio version 1.0.153. A file is transmitted in the Supporting Information with all R codes and the packages used S1 R Codes .", "Statistical analyses were done using R for Mas OS X version R 3.4.1 software with RStudio version 1.0.153. A file is transmitted in the Supporting Information with all R codes and the packages used S1 R Codes . Differences were considered significant at p � 0.05. The French Data Protection Authority approved data collection and analysis DE-2013-074 and the local ethics committee Espace Local de Réflexion Ethique, Centre Hospitalier de Rouffach approved the study protocol ERLE-32 . According to the French law for biomedical research and human experimentation, individual written consent was not required from the patients or their relatives for data collection. Each year, the referring local practitioner of the study coordinated with the doctors working in the nursing home.", "According to the French law for biomedical research and human experimentation, individual written consent was not required from the patients or their relatives for data collection. Each year, the referring local practitioner of the study coordinated with the doctors working in the nursing home. At the beginning of the surveillance period, information regarding participation in the study was displayed in the family vising area, including a document about their right to access and rectify personal data. After collection, data were rendered anonymous. No specific authorization was needed to retrospectively analyze anonymous data collected during routine care in the context of routine surveillance. A total of 137 outbreaks were recorded in the 14 sites.", "No specific authorization was needed to retrospectively analyze anonymous data collected during routine care in the context of routine surveillance. A total of 137 outbreaks were recorded in the 14 sites. RTI outbreaks were more frequent than GE outbreaks 76 outbreaks and 5862 exposed residents vs. 61 outbreaks and 4309 exposed residents, respectively . Overall, 7643 of the exposed residents were women and 2528 were men. The median age was 86.7 years old interquartile range: 81.1-91.0 years . Virus investigations respectively 389 samples for RTI and 143 for GE with all the detailed results in S1-S4 Tables confirmed a considerable number of norovirus-related GE outbreaks 34/61 and influenza-related RTI outbreaks 46/76 .", "The median age was 86.7 years old interquartile range: 81.1-91.0 years . Virus investigations respectively 389 samples for RTI and 143 for GE with all the detailed results in S1-S4 Tables confirmed a considerable number of norovirus-related GE outbreaks 34/61 and influenza-related RTI outbreaks 46/76 . For GE outbreaks, 2524 residents were in a NoV+ context versus 1785 in a NoV-context, and for RTI outbreaks, 3479 residents were in a Flu+ context versus 2383 in a Flu-context. For GE surveillance in the NoV+ context, there were 1093 EI residents versus 1431 ENI residents, whereas in the NoV-context, there were 583 EI residents versus 1202 ENI residents. Therefore, the infection rate was higher in the NoV+ context 43.3% than in the NoV-context 32.7%, odds ratio OR : 0.63, 95% confidence interval CI : 0.56-0.72 , p < 0.001 . For RTI surveillance, the rates of infection were similar with and without confirmed influenza: 31.5% N = 1095 EI residents /3479 exposed residents vs. 30.5% N = 728 EI residents/ 2383 exposed residents, OR: 0.96, CI: 0.85-1.07, p = 0.47 .", "Therefore, the infection rate was higher in the NoV+ context 43.3% than in the NoV-context 32.7%, odds ratio OR : 0.63, 95% confidence interval CI : 0.56-0.72 , p < 0.001 . For RTI surveillance, the rates of infection were similar with and without confirmed influenza: 31.5% N = 1095 EI residents /3479 exposed residents vs. 30.5% N = 728 EI residents/ 2383 exposed residents, OR: 0.96, CI: 0.85-1.07, p = 0.47 . Moreover, infection rate in the NoV + context was higher than the three other contexts: NoV- OR: 0.63, CI: 0.56-0.72 , Flu+ OR: 0.60, CI:0.54-0.67 and Flu- OR: 0.58, CI: 0.51-0.65 . In univariate analysis, certain individual characteristics were associated with significant variations in the infection rate S5 Table . The infection rate increased with age except in the Flucontext and, decreased with LOS during GE outbreaks. The covariate adjusted analysis revealed specific significant effect modification according to sex NoV+ and LOS NoV- S6 Table .", "The infection rate increased with age except in the Flucontext and, decreased with LOS during GE outbreaks. The covariate adjusted analysis revealed specific significant effect modification according to sex NoV+ and LOS NoV- S6 Table . In analyses stratified according to virus and sex, age adjusted for LOS remained significant for Flu+ and NoV+ outbreaks males . In NoV-context, the effect modification of LOS remained significant Table 1 . Finally, when autonomy was included and adjusted for age virus, sex, LOS stratification , the less autonomous residents female/LOS<4 years/age<86/ GIR 1-2 were affected more severely by Flu+ outbreaks with specific effect modification according to age S7 Table . The study of lethality rates in infected residents over the 56 days after onset indicated that there were significant variations for RTI but no change for GE Table 2 .", "Finally, when autonomy was included and adjusted for age virus, sex, LOS stratification , the less autonomous residents female/LOS<4 years/age<86/ GIR 1-2 were affected more severely by Flu+ outbreaks with specific effect modification according to age S7 Table . The study of lethality rates in infected residents over the 56 days after onset indicated that there were significant variations for RTI but no change for GE Table 2 . Significant differences appeared after 28 days in the context of Flu+ outbreaks and other RTI outbreaks. The analysis of successive or simultaneous clusters in the same institutions was performed when the time period between the onset of symptoms of the last case in outbreak N and the onset of symptoms of the first case in outbreak N+1 was �56 days S8 Table . 44 of the 137 outbreaks 32.12% were identified, and 194 of the 3499 exposed and infected residents contracted multiple infections. The percentage of exposed and infected residents implicated in more than one virus stratification was 11.09% 194 � 2 /3499 .", "44 of the 137 outbreaks 32.12% were identified, and 194 of the 3499 exposed and infected residents contracted multiple infections. The percentage of exposed and infected residents implicated in more than one virus stratification was 11.09% 194 � 2 /3499 . Moreover, two deceased residents were included in the NoV-Na and Flu lethality analyses because death occurred within 56 days for both infections. The analysis of virus stratification of the 44 outbreaks showed the absence of successive clusters for the same category. The same analysis for the first four 7-day intervals Days 1 to 28 showed the respective values: 26 outbreaks 18.98% , 117 residents 6.69% 117 � 2 /3499 , one dead resident. Finally, according to the higher lethality impact during the first four 7-day intervals and to limit the impact of successive clusters in the same site, all cause lethality rates were studied according to individual parameters for the four 7 days intervals with the respective number of According to the surveillance type GE or RTI , the lethality rates differed significantly: 1.6% versus 3.4% respectively NoV+ and NoV-contexts, OR: 2.24, CI: 1.16-4.39, p = 0.02 and 8.3% versus 5.6% respectively Flu+ and Flu-, OR: 0.67, CI: 0.45-0.97, p = 0.04 .", "The same analysis for the first four 7-day intervals Days 1 to 28 showed the respective values: 26 outbreaks 18.98% , 117 residents 6.69% 117 � 2 /3499 , one dead resident. Finally, according to the higher lethality impact during the first four 7-day intervals and to limit the impact of successive clusters in the same site, all cause lethality rates were studied according to individual parameters for the four 7 days intervals with the respective number of According to the surveillance type GE or RTI , the lethality rates differed significantly: 1.6% versus 3.4% respectively NoV+ and NoV-contexts, OR: 2.24, CI: 1.16-4.39, p = 0.02 and 8.3% versus 5.6% respectively Flu+ and Flu-, OR: 0.67, CI: 0.45-0.97, p = 0.04 . In univariate analysis S9 Table , low autonomy status in the NoV+, Flu+ and Flu-contexts was most significantly associated with increased all-cause lethality, and age was associated with higher lethality in the Flu+ context. In the adjusted analysis, no significant statistical differences were identified in GE outbreaks. For RTI episodes, the adjusted analysis showed that autonomy had a significant impact when adjusted for sex, age or LOS Flu+ and Flu-NA and that age had a significant impact when adjusted for sex, autonomy or LOS Flu+ S10 Table . In Table 3 , the specific effects of age or autonomy were tested.", "For RTI episodes, the adjusted analysis showed that autonomy had a significant impact when adjusted for sex, age or LOS Flu+ and Flu-NA and that age had a significant impact when adjusted for sex, autonomy or LOS Flu+ S10 Table . In Table 3 , the specific effects of age or autonomy were tested. Significant OR age adjusted for autonomy were: Flu+/age �86 years compared with the <86 group , 1.97 1.19-3.25 . OR autonomy adjusted for age were for GIR 3-6 compared with GIR 1-2 : Flu+, 0.41 0.24-0.69 ; Flu-, 0.42 0.20, 0.90 . Finally, despite the low number of residents and deaths per category, and consequently the limited robustness of the results, autonomy adjusted for age with stratification according to virus, sex and LOS showed that the effects were higher among subgroups of less autonomous residents female or male/LOS<4 years/GIR 1-2 in Flu+ outbreaks, and there was also higher mortality in the small subgroup of autonomous men with LOS � 4 years higher mortality S11 Table . In the Flu+ context, data regarding vaccination status and oseltamivir prescriptions were available but not used in this study.", "Finally, despite the low number of residents and deaths per category, and consequently the limited robustness of the results, autonomy adjusted for age with stratification according to virus, sex and LOS showed that the effects were higher among subgroups of less autonomous residents female or male/LOS<4 years/GIR 1-2 in Flu+ outbreaks, and there was also higher mortality in the small subgroup of autonomous men with LOS � 4 years higher mortality S11 Table . In the Flu+ context, data regarding vaccination status and oseltamivir prescriptions were available but not used in this study. In the present study, surveillance data obtained during GE and RTI outbreaks in nursing homes were used to construct stratified analyses and to identify specific infection and all-cause lethality rates according to the residents' individual characteristics. The infection rates observed here were similar to those found in previous studies of NoV and Influenza outbreaks odds of being infected during a Flu+ outbreak were around 40% less than during a NoV+ outbreak . Reported infection rates were close to 30.0% in influenza outbreaks and 40.0% in NoV outbreaks . Older age appeared to increase the likelihood of GE and influenza infection, with increasing rates among older residents.", "Reported infection rates were close to 30.0% in influenza outbreaks and 40.0% in NoV outbreaks . Older age appeared to increase the likelihood of GE and influenza infection, with increasing rates among older residents. Age is a well-known factor for influenza and norovirus severity in the elderly and in nursing homes . For NoV, the highest incidence estimates 5-year age strata was found in the �85 year-category approximately 800 men and for 1,400 women per 100,000 inhabitants . In our study, univariate analysis NoV+ showed that the odds of being infected were 1.5 to 1.6 times higher if a resident was older than 85. Moreover, an adjusted analysis of GE outbreaks highlighted different effects among subgroups of residents according to sex and LOS.", "In our study, univariate analysis NoV+ showed that the odds of being infected were 1.5 to 1.6 times higher if a resident was older than 85. Moreover, an adjusted analysis of GE outbreaks highlighted different effects among subgroups of residents according to sex and LOS. Indeed, multiple and/or repeated exposure to GE viruses while institutionalized may lead to susceptibility or possible increased immunity in some residents . For the sex variable, two factors could explain the effect: a possible selection bias with men reporting mild infections less than women particularly in the <86 years subgroup or that male susceptibility was different age, LOS, immunity,. . . .", ". . . A German study from 2013 also reported a greater impact in women . Moreover, when age analysis was stratified by sex and LOS, no Epidemic impacts in nursing homes significant impact was observed in women; the only significant differences were fewer infections in men in the <86-subgroup except in the NoV-with LOS <4 years . For the RTI outbreaks, sex and LOS variables did not have a significant effect. In residents older than 86, the odds of being infected in Flu+ context were 1.5 times higher for women and 1.7 for men.", "For the RTI outbreaks, sex and LOS variables did not have a significant effect. In residents older than 86, the odds of being infected in Flu+ context were 1.5 times higher for women and 1.7 for men. In univariate analysis, contrary to the other virus contexts where odds ratios were rarely above 2, residents over 95 years old had increased odds of infection of � 2.8 compared with the 70-year-old category, and for the 100 year-old group the odds were approximately 3.8. In the Flu+ context, autonomy adjusted for age virus, sex and LOS stratification revealed a possible increase in infection rates among less autonomous residents. A previous study found that when elderly residents were exposed to the A H3N2 virus, there were higher rates of infection and reinfection, and more significant effects on the institution than with other influenza types/subtypes. In the community, the relative illness ratio RIR in the .", "A previous study found that when elderly residents were exposed to the A H3N2 virus, there were higher rates of infection and reinfection, and more significant effects on the institution than with other influenza types/subtypes. In the community, the relative illness ratio RIR in the . The incidence of influenza infection and the associated risks were well described by age group, but the specific impact according to age was not studied. The results of this work highlighted the specific age distribution of influenza illnesses among the nursing home residents and the more significant impact among the older residents. This specific susceptibility could be a critical factor in the institutional exposure and dissemination of influenza and could partly explain the high infection impact in the elderly institutional population. In this work, autonomy status was not the main factor associated with infection no significant impact in GE and in Flu-contexts .", "This specific susceptibility could be a critical factor in the institutional exposure and dissemination of influenza and could partly explain the high infection impact in the elderly institutional population. In this work, autonomy status was not the main factor associated with infection no significant impact in GE and in Flu-contexts . However, in Flu+ outbreaks, a high level of dependency was associated with a higher risk of falling ill. This observation implies that staff could play a role in the spread of infection highly dependent and less mobile residents are less likely to contaminate themselves or that the more active residents may be less fragile and/or have a greater involvement in the recommended infection control measures. Finally, improving compliance with personal hygiene measures both for nursing staff and residents might be expected to have a beneficial effect on infection rates. Previous studies identified higher NoV infection rates in highly dependent individuals, but the results were not adjusted for age and LOS to take into account the potential correlation with the autonomy status .", "Finally, improving compliance with personal hygiene measures both for nursing staff and residents might be expected to have a beneficial effect on infection rates. Previous studies identified higher NoV infection rates in highly dependent individuals, but the results were not adjusted for age and LOS to take into account the potential correlation with the autonomy status . Lethality is difficult to assess in nursing homes because death is frequent. Our GE and RTI episodes occurred during the winter seasons, and there are possible interactions between outbreaks and increased mortality at this time of the year . The all-cause lethality rate of the infected residents in our study reflected global mortality including GE and RTI outbreaks and the global epidemiological context. Not surprisingly, a higher all-cause lethality rate was observed in the influenza contexts, as reported in previous studies .", "The all-cause lethality rate of the infected residents in our study reflected global mortality including GE and RTI outbreaks and the global epidemiological context. Not surprisingly, a higher all-cause lethality rate was observed in the influenza contexts, as reported in previous studies . Age and autonomy had similar effects in the different contexts, but in GE and to a lesser degree in Flu-outbreaks, the relatively small number of deaths could have limited the power of the statistical tests. In nursing homes, residents are generally discharged due to death. The number of residents lost to follow up was low 0.14% in the first 28 days , so the 7-day interval turnover rate calculated on the base of the median LOS provides a good indication of the average case fatality rate. The lethality rate for NoV+ outbreaks was similar to the estimated 7-day interval turnover rate 1.6% indicating that this context had a limited impact on the death rate.", "The number of residents lost to follow up was low 0.14% in the first 28 days , so the 7-day interval turnover rate calculated on the base of the median LOS provides a good indication of the average case fatality rate. The lethality rate for NoV+ outbreaks was similar to the estimated 7-day interval turnover rate 1.6% indicating that this context had a limited impact on the death rate. The all-cause lethality rate was most affected by age and autonomy. Both individual characteristics were significant in the Flu+ outbreaks, and autonomy adjusted for age was significant in the Flu-episodes. The influence of age on mortality in a context of influenza has already been described: a very high mortality rate 831/100,000 inhabitants was reported in persons 90 years of age and older compared with those aged 65-69 years 23/100,000 inhabitants . In our univariate analysis, the higher risk was observed in the �90 group whose risk of death was at least 2.6 higher than the <70 group.", "The influence of age on mortality in a context of influenza has already been described: a very high mortality rate 831/100,000 inhabitants was reported in persons 90 years of age and older compared with those aged 65-69 years 23/100,000 inhabitants . In our univariate analysis, the higher risk was observed in the �90 group whose risk of death was at least 2.6 higher than the <70 group. When adjusted for autonomy, the impact of age was not significant in more autonomous residents in the Flu+ context and not at all in the Flucontext. The opposite analysis autonomy adjusted for age showed higher global impact in the less autonomous group Flu- or only in the �86 age group Flu+ . Age and autonomy are a reflection of resident's level of frailty. Clinical frailty scores were not used in this study, but in a previous study of patients with critical illness, they were associated with greater mortality, regardless of age .", "Age and autonomy are a reflection of resident's level of frailty. Clinical frailty scores were not used in this study, but in a previous study of patients with critical illness, they were associated with greater mortality, regardless of age . This suggests that in addition to age, autonomy can be a valuable indicator for the assessment of outbreak impact in outbreak surveillance. Other studies have suggested that age and certain comorbidities are independent risk factors for the influenza mortality rate or that mortality increase according to the number of risk factors . Comorbidities and underlying diseases of various severities could reflect overall frailty and consequently the risk of death. In nursing homes, information about autonomy and age are easier to collect and interpret than data on comorbidities.", "Comorbidities and underlying diseases of various severities could reflect overall frailty and consequently the risk of death. In nursing homes, information about autonomy and age are easier to collect and interpret than data on comorbidities. These various approaches should be evaluated and compared in the goal of optimizing risk assessment among nursing home residents. The present work has two main limitations. First, the virus information was incomplete limited identification, mainly influenza rapid tests for the RTI . Consequently, some episodes in the levels with no available identification may also have been associated with influenza or norovirus, and multiple contaminations could have been underestimated or not taken into account.", "First, the virus information was incomplete limited identification, mainly influenza rapid tests for the RTI . Consequently, some episodes in the levels with no available identification may also have been associated with influenza or norovirus, and multiple contaminations could have been underestimated or not taken into account. Moreover, vaccination and oseltamivir prescriptions were recorded but not included because the influenza genotype was not determined and identification was limited. Secondly, the deaths of uninfected residents were not recorded in this protocol even though such data would have provided valuable information about the global epidemiological context. In conclusion, specific susceptibility patterns were observed among exposed residents. In this cohort of nursing homes, infection rates varied according to virus, sex, length of stay and age, and there were major differences in lethality depending on virus, age and autonomy score.", "In conclusion, specific susceptibility patterns were observed among exposed residents. In this cohort of nursing homes, infection rates varied according to virus, sex, length of stay and age, and there were major differences in lethality depending on virus, age and autonomy score. The collected data were easy to record and could be used to improve the characterization of seasonal outbreaks in nursing homes, whose residents are particularly vulnerable. Finally, as the average age and dependency level of residents continues to increase, subsequently increasing the risk of infection and death, health care staff will have to be increasingly vigilant during seasonal outbreaks and targeted interventions should be implemented. Supporting information S1 Data. CSV S1 R Codes.", "Supporting information S1 Data. CSV S1 R Codes. R S1 Table. XLSX S10 Table. XLSX S11 Table. XLSX" ]

[ "BACKGROUND: Gastroenteritis GE and respiratory tract infection RTI outbreaks are a significant issue in nursing homes. This study aimed to describe GE and RTI outbreaks with infection and all-cause lethality rates according to the individual characteristics of nursing home residents. METHODS: Clinical and virological surveillance were conducted 2007 to 2018 . Virus stratifications for the analysis were: outbreaks with positive norovirus or influenza identifications respectively NoV+ or Flu+ , episodes with no NoV or influenza identification or testing respectively NoV- or Flu- . Associations between individual variables sex, age, length of stay LOS , autonomy status and infection and lethality rates were tested with univariate and Mantel-Haenszel MH methods. RESULTS: 61 GE outbreaks and 76 RTI oubreaks total 137 outbreaks were recorded involving respectively 4309 and 5862 residents.", "Associations between individual variables sex, age, length of stay LOS , autonomy status and infection and lethality rates were tested with univariate and Mantel-Haenszel MH methods. RESULTS: 61 GE outbreaks and 76 RTI oubreaks total 137 outbreaks were recorded involving respectively 4309 and 5862 residents. In univariate analysis, higher infection rates and age were associated in NoV+, NoV-, and Flu+ contexts, and lower infection rates were associated with longer stays NoV+ and NoV- . In MH stratified analysis virus, sex female/male adjusted for LOS <4 or ≥4 years , the odds of being infected remained significant among older residents ≥86 years : NoV+/male Odds ratio OR MH : 1.64, 95% confidence interval CI : 1.16–2.30 and Flu+/female and male respectively OR MH : 1.50, CI: 1.27–1.79 and 1.73, CI: 1.28–2.33 . In univariate analysis, lower autonomy status NoV+, Flu+ and Flu- and increased age Flu+ were associated with higher lethality. In MH adjusted analysis, significant OR age adjusted for autonomy was: Flu+/ ≥86 years compared with <86 years, 1.97 1.19–3.25 and OR autonomy adjusted for age for the more autonomous group compared with the less autonomous group was: Flu+, 0.41 0.24–0.69 ; Flu-, 0.42 0.20, 0.90 .", "In univariate analysis, lower autonomy status NoV+, Flu+ and Flu- and increased age Flu+ were associated with higher lethality. In MH adjusted analysis, significant OR age adjusted for autonomy was: Flu+/ ≥86 years compared with <86 years, 1.97 1.19–3.25 and OR autonomy adjusted for age for the more autonomous group compared with the less autonomous group was: Flu+, 0.41 0.24–0.69 ; Flu-, 0.42 0.20, 0.90 . CONCLUSION: The residents of nursing homes are increasingly elderly and dependent. The specific infection and lethality risks according to these two factors indicate that surveillance and infection control measures are essential and of high priority. Text: Introduction Gastroenteritis GE and respiratory tract infection RTI outbreaks represent a significant burden of illness in nursing homes. Viruses cause the majority of these outbreaks, and noroviruses and influenza viruses are the most common pathogens .", "Text: Introduction Gastroenteritis GE and respiratory tract infection RTI outbreaks represent a significant burden of illness in nursing homes. Viruses cause the majority of these outbreaks, and noroviruses and influenza viruses are the most common pathogens . Previous studies have suggested that viral respiratory infections and norovirus outbreaks are a common cause of hospitalization or death, particularly among elderly individuals . The impact of outbreaks has been described in terms of both frequency and epidemiology, but little is known about infection rates and all-cause lethality in GE and RTI nursing home outbreaks in relation to the individual characteristics of the residents . The residents of these institutions are increasingly elderly and dependent, and the impact of this trend on the seasonal outbreak burden requires in-depth investigation. The results of studies focused on this issue could yield valuable information for nursing homes, allowing them to adapt their infection control strategies, in particular for improved assessment of infection risk.", "The residents of these institutions are increasingly elderly and dependent, and the impact of this trend on the seasonal outbreak burden requires in-depth investigation. The results of studies focused on this issue could yield valuable information for nursing homes, allowing them to adapt their infection control strategies, in particular for improved assessment of infection risk. Our objective was to describe GE and RTI infection and all-cause lethality rates according to the individual characteristics of nursing home residents sex, age, length of stay, autonomy status , and to identify specific susceptibility patterns related to these types of viral outbreaks in these facilities. The present study explored outbreaks in 14 sites 28 units with geriatric nursing home activities for a total of 1121 beds caring for dependent people in southern Alsace an area in northeastern France . Data were collected between September 2007 and August 2018 . Each site was geographically independent and autonomous for social and care management.", "Data were collected between September 2007 and August 2018 . Each site was geographically independent and autonomous for social and care management. Units were located within the larger sites and were defined as a place having a dedicated team at one location. During outbreaks at one site, only the residents in the units with confirmed cases were included. Outbreak inclusion depended on institutional alert to the hygiene team. Surveillance was done in each unit independently, and the members of staff had to inform a physician or charge nurse when two or more potential related cases of pneumonia or GE were observed within four days and when three or more cases were observed for other RTI.", "Outbreak inclusion depended on institutional alert to the hygiene team. Surveillance was done in each unit independently, and the members of staff had to inform a physician or charge nurse when two or more potential related cases of pneumonia or GE were observed within four days and when three or more cases were observed for other RTI. Units also had to inform the hygiene team when these threshold values were exceeded. For influenza, the first suspected case led to a local alert and the hygiene team was contacted. A practitioner from the hygiene team collected the information and evaluated the clinical signs, the virology information and the epidemiological context with the physician in the affected unit. The detected cluster was only put under surveillance if the hygiene team considered that there was a potential outbreak phenomenon.", "A practitioner from the hygiene team collected the information and evaluated the clinical signs, the virology information and the epidemiological context with the physician in the affected unit. The detected cluster was only put under surveillance if the hygiene team considered that there was a potential outbreak phenomenon. The duration of 4 days was in relation with the national protocol with alert to the authorities when 5 cases occurred within 4 days . On a local level and in addition to the clusters reported to the authorities, clusters with at least 3 cases within a period of seven days in one unit could be recorded if they were reported to the hygiene team. Because several outbreaks could potentially occur in the same unit during the surveillance period, a resident could be included repeatedly in different clusters. As a result, the observed patterns reflected the characteristics of an institutional population with longitudinal and pluriannual exposures.", "Because several outbreaks could potentially occur in the same unit during the surveillance period, a resident could be included repeatedly in different clusters. As a result, the observed patterns reflected the characteristics of an institutional population with longitudinal and pluriannual exposures. Personal information and clinical information was collected by a practitioner from the hygiene team directly from the residents' health care records. Personal information was collected for all those present the first day of the outbreak. The collected information included: month and year of birth, sex, date of arrival at the nursing home and autonomy status. The autonomy status of residents in French nursing homes is assessed using the AGGIR scale Autonomy Gerontology Groups Iso-Resources , which is the legal instrument for evaluating dependency in the elderly and whose primary purpose is the allocation of means and resources .", "The collected information included: month and year of birth, sex, date of arrival at the nursing home and autonomy status. The autonomy status of residents in French nursing homes is assessed using the AGGIR scale Autonomy Gerontology Groups Iso-Resources , which is the legal instrument for evaluating dependency in the elderly and whose primary purpose is the allocation of means and resources . With the AGGIR scale, autonomy is classified into 6 Iso-Resource Groups GIR : GIR 1 bedridden or armchair-bound persons, mental functions seriously altered and requiring continuous presence , GIR 2 bedridden or armchair-bound persons, mental functions not totally altered and requiring assistance in most activities of daily living, or mental functions altered with preserved ability to get around , GIR 3 preserved mental autonomy with partially preserved motor autonomy and assistance several times a day for physical autonomy , GIR 4 moves around the home and sometimes assistance for washing, dressing, physical activities or eating , GIR 5 only occasional assistance for washing, meal preparation, and housework , GIR 6 autonomy for essential tasks of daily living . In outbreaks where the influenza virus was identified, influenza vaccination status and oseltamivir prescriptions were recorded as well. A file is transmitted in the Supporting Information with all previous data S1 Data . GE was defined as the sudden onset of vomiting and/or diarrhea over a 24 h period: i diarrhea �3 episodes, ii and/or vomiting �3 episodes, iii or diarrhea or vomiting <3 episodes with two or more other symptoms diarrhea, vomiting, stomach ache, abdominal cramps, nausea, fever, mucus in stools .", "A file is transmitted in the Supporting Information with all previous data S1 Data . GE was defined as the sudden onset of vomiting and/or diarrhea over a 24 h period: i diarrhea �3 episodes, ii and/or vomiting �3 episodes, iii or diarrhea or vomiting <3 episodes with two or more other symptoms diarrhea, vomiting, stomach ache, abdominal cramps, nausea, fever, mucus in stools . RTI presentation in older adults may be atypical, like for other acute illnesses in this age group . We used the recommended definitions for RTI surveillance in geriatric units, divided in 3 subcategories: i common cold syndromes or pharyngitis at least two of the following criteria: runny nose or sneezing, stuffy nose i.e. congestion , sore throat or hoarseness or difficulty swallowing, dry cough, swollen or tender glands in the neck cervical lymphadenopathy , ii influenza-like illness both the following criteria must be met: fever AND at least three other symptoms chills, new headache or eye pain, myalgia or body aches, malaise or loss of appetite, sore throat, new or increased dry cough and iii lower respiratory tract infection both of the following criteria must be met: at least two respiratory signs or symptoms new or increased cough, new/increased sputum production, O 2 saturation <94% or reduced >3% from baseline, abnormal lung examination new or changed , pleuritic chest pain, respiratory rate �25 breaths/min AND one or more constitutional signs/symptoms fever, leukocytosis, confusion, acute functional decline . Infection corresponding to one of these three subcategories was included in this study and classified as RTI.", "congestion , sore throat or hoarseness or difficulty swallowing, dry cough, swollen or tender glands in the neck cervical lymphadenopathy , ii influenza-like illness both the following criteria must be met: fever AND at least three other symptoms chills, new headache or eye pain, myalgia or body aches, malaise or loss of appetite, sore throat, new or increased dry cough and iii lower respiratory tract infection both of the following criteria must be met: at least two respiratory signs or symptoms new or increased cough, new/increased sputum production, O 2 saturation <94% or reduced >3% from baseline, abnormal lung examination new or changed , pleuritic chest pain, respiratory rate �25 breaths/min AND one or more constitutional signs/symptoms fever, leukocytosis, confusion, acute functional decline . Infection corresponding to one of these three subcategories was included in this study and classified as RTI. For both infection types, the practitioner from the hygiene team obtained clinical information from the patient's health care records, and members of the health care team were consulted if necessary to complete any missing information. At the end of the episode within seven days after the last identified case , case inclusion as exposed and not infected ENI or exposed and infected EI was determined with a resident physician. In order to study the lethality, the presence of each infected resident was evaluated once at least 56 days after the last case of each outbreak. Each resident was followed up retrospectively during eighth 7-day interval I n, n = 1 to 8, total 56 days, between the date of onset of symptoms and the fifty-sixth day of the studied period with three different possibilities: present alive and officially residing in the institution , lost to follow-up alive at the date of departure but no longer residing in the institution return home, transfer to another institution or death death recorded in the health care record .", "In order to study the lethality, the presence of each infected resident was evaluated once at least 56 days after the last case of each outbreak. Each resident was followed up retrospectively during eighth 7-day interval I n, n = 1 to 8, total 56 days, between the date of onset of symptoms and the fifty-sixth day of the studied period with three different possibilities: present alive and officially residing in the institution , lost to follow-up alive at the date of departure but no longer residing in the institution return home, transfer to another institution or death death recorded in the health care record . The dates of death and lost to follow-up were recorded. Testing for the virus was not systematic and was decided by the physicians in each institution in the presence of clinical signs. For GE, stool samples were sent to the National Reference Centre for Gastroenteritis Viruses in Dijon for laboratory testing, as previously described . For RTI surveillance, rapid tests were used to identify the influenza virus.", "For GE, stool samples were sent to the National Reference Centre for Gastroenteritis Viruses in Dijon for laboratory testing, as previously described . For RTI surveillance, rapid tests were used to identify the influenza virus. The rapid immunoassay diagnosis tests used for influenza detection were: Clearview1 Exact Influenza A and B Inverness Medical, Cologne, Germany from 2007 to 2014 and InfluenzaTop1 Alldiag, Strasbourg, France from 2014 to 2018. Given the low sensitivity of influenza rapid tests, they were no longer used once the control measures had been implemented and the influenza outbreak was under control. Samples were also occasionally sent to hospital laboratories or to the National Reference Centre for Influenza Viruses to detect viruses with real-time RT-PCR . Most testing targeted the norovirus NoV and influenza virus, but other tests were occasionally performed by the National Reference Centre for Influenza Viruses rhinovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza 1, 2, 3 and 4, and coronavirus and the National Reference Centre for Enteric Viruses rotavirus, astrovirus, and adenovirus .", "Samples were also occasionally sent to hospital laboratories or to the National Reference Centre for Influenza Viruses to detect viruses with real-time RT-PCR . Most testing targeted the norovirus NoV and influenza virus, but other tests were occasionally performed by the National Reference Centre for Influenza Viruses rhinovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza 1, 2, 3 and 4, and coronavirus and the National Reference Centre for Enteric Viruses rotavirus, astrovirus, and adenovirus . Because testing for the viruses was variable from one institution/physician to another, not used in some outbreaks, types of virus sought and considering the poor sensitivity of the rapid influenza tests, these two sources of data were used to define the epidemiological context of confirmed outbreaks. Consequently, individual cases were included consistently in all episodes according to clinical signs and medical evaluation. When virus testing was negative, the clinical signs were recorded and medical evaluation was used as previously to classify the included residents as infected or not infected. The epidemiological context of each outbreak was defined according to whether the virus had been identified or not.", "When virus testing was negative, the clinical signs were recorded and medical evaluation was used as previously to classify the included residents as infected or not infected. The epidemiological context of each outbreak was defined according to whether the virus had been identified or not. One or more positive samples led to the qualification of a NoV NoV+ or flu Flu+ context. The other episodes were qualified as flu or NoV outbreaks with no specific identification or testing NoV-and Flu- . Flu and NoV contexts did not eliminate other potential enteric or respiratory pathogens. Sex, age, length of stay LOS, in years and autonomy status were described for all exposed residents.", "Flu and NoV contexts did not eliminate other potential enteric or respiratory pathogens. Sex, age, length of stay LOS, in years and autonomy status were described for all exposed residents. Influenza vaccination and oseltamivir administration rates were calculated for confirmed influenza outbreaks. Dichotomous or categorical variables were expressed as percentages. In univariate analysis, the categories were specific in order to obtain a precise description of the age and LOS variables. Class intervals were 5 years for age and one year for LOS. The residents classified as GIR 4 to 6 sometimes, occasional and no assistance were grouped together because they were few.", "Class intervals were 5 years for age and one year for LOS. The residents classified as GIR 4 to 6 sometimes, occasional and no assistance were grouped together because they were few. For the multi-level analysis with 2x2 tables, a median value was used to define the two-level age categories. For LOS, assessing the longest stays was necessary to identify the effect of longer exposure in a nursing home. Consequently, a four-year cutoff was chosen to create the two categories. For autonomy, the two most dependent categories GIR � 2 were grouped together and compared with the more autonomous categories GIR � 3 .", "Consequently, a four-year cutoff was chosen to create the two categories. For autonomy, the two most dependent categories GIR � 2 were grouped together and compared with the more autonomous categories GIR � 3 . The outbreak epidemiological contexts were used with the four categories: NoV+, Flu+, NoV-and flu-. Other GE or RTI viruses were occasionally identified, but the number of results was too limited to develop separate analyses. However, all the results are available in the tables about the virus investigations along with NoV and influenza identifications. For the different categories, infection rate EI/Exposed Residents ER , in percentage was calculated according to sex, age group, LOS and autonomy status.", "However, all the results are available in the tables about the virus investigations along with NoV and influenza identifications. For the different categories, infection rate EI/Exposed Residents ER , in percentage was calculated according to sex, age group, LOS and autonomy status. To investigate all-cause lethality and define the appropriate period for the 56-day monitoring D 1 to 56 , the all-cause lethality rate per 7-day interval LR n /I n, n = 1 to 8 was calculated: number of deaths during interval I n / EI alive the first day of n th studied interval minus lost to follow-up EI during the interval I n � 100 . Seeing as successive clusters could occur within the same site, potentially influencing allcause lethality, the serial interval in days SI d was calculated. The SI d was the time period between the onset of symptoms of the last case in initial outbreak N and the onset of symptoms of the first case in the following outbreak N+1 . Investigations were performed when SI d was shorter or equal to the length of the previous D 1 to 56 and the following parameters were evaluated for these specific situations: number of episodes, residents infected in both outbreaks, and death among the identified individuals.", "The SI d was the time period between the onset of symptoms of the last case in initial outbreak N and the onset of symptoms of the first case in the following outbreak N+1 . Investigations were performed when SI d was shorter or equal to the length of the previous D 1 to 56 and the following parameters were evaluated for these specific situations: number of episodes, residents infected in both outbreaks, and death among the identified individuals. Finally, according to the death rate and the impact of successive outbreaks, the number of 7-day intervals N.I n to take into account was defined, and the all-cause lethality rate was analyzed during these periods I 1 to N th .I n or D 1 to 7 � N . All-cause lethality rates were calculated with the following formula: number of deaths from D 1 to 7 � N / EI number at D 1 minus lost to follow-up among EI during the period D 1 to 7 � N � 100 . Estimation of the turnover rate per 7-day interval among the infected residents was calculated on the base of the LOS median in years with the following formula: proportion of discharged residents: 50.0% in the case of the median / median LOS � 365 /7 . The average rate of residents discharged per 7-day period was calculated: number of lost to follow up during the period D 1 to 7 � N /number of exposed and infected residents at D 1 /N 7-day interval � 100.", "Estimation of the turnover rate per 7-day interval among the infected residents was calculated on the base of the LOS median in years with the following formula: proportion of discharged residents: 50.0% in the case of the median / median LOS � 365 /7 . The average rate of residents discharged per 7-day period was calculated: number of lost to follow up during the period D 1 to 7 � N /number of exposed and infected residents at D 1 /N 7-day interval � 100. As some residents were included in several outbreaks during the surveillance, the observations were not completely independent; non-parametric tests were used as a result. In univariate analysis, Chi-square or Fisher exact tests expected number of frequencies fewer than 5 were used to compare infection and lethality rates according to the studied parameters and the odds ratio was calculated by median-unbiased estimation. The Kruskal-Wallis test was used to compare median values. Confidence intervals for medians were calculated with bootstrap methods.", "The Kruskal-Wallis test was used to compare median values. Confidence intervals for medians were calculated with bootstrap methods. Covariate adjusted analyses were performed with two tables 2x2 . The respective impact of each individual factor was tested with Mantel-Haenszel chi-squared tests. The equality of the stratum odds ratios was tested with the Woolf test of homogeneity. Finally, for each virus context, multiple tables 2x2 were generated and tested with confounding variables, effect modifiers or covariables. Statistical analyses were done using R for Mas OS X version R 3.4.1 software with RStudio version 1.0.153. A file is transmitted in the Supporting Information with all R codes and the packages used S1 R Codes .", "Statistical analyses were done using R for Mas OS X version R 3.4.1 software with RStudio version 1.0.153. A file is transmitted in the Supporting Information with all R codes and the packages used S1 R Codes . Differences were considered significant at p � 0.05. The French Data Protection Authority approved data collection and analysis DE-2013-074 and the local ethics committee Espace Local de Réflexion Ethique, Centre Hospitalier de Rouffach approved the study protocol ERLE-32 . According to the French law for biomedical research and human experimentation, individual written consent was not required from the patients or their relatives for data collection. Each year, the referring local practitioner of the study coordinated with the doctors working in the nursing home.", "According to the French law for biomedical research and human experimentation, individual written consent was not required from the patients or their relatives for data collection. Each year, the referring local practitioner of the study coordinated with the doctors working in the nursing home. At the beginning of the surveillance period, information regarding participation in the study was displayed in the family vising area, including a document about their right to access and rectify personal data. After collection, data were rendered anonymous. No specific authorization was needed to retrospectively analyze anonymous data collected during routine care in the context of routine surveillance. A total of 137 outbreaks were recorded in the 14 sites.", "No specific authorization was needed to retrospectively analyze anonymous data collected during routine care in the context of routine surveillance. A total of 137 outbreaks were recorded in the 14 sites. RTI outbreaks were more frequent than GE outbreaks 76 outbreaks and 5862 exposed residents vs. 61 outbreaks and 4309 exposed residents, respectively . Overall, 7643 of the exposed residents were women and 2528 were men. The median age was 86.7 years old interquartile range: 81.1-91.0 years . Virus investigations respectively 389 samples for RTI and 143 for GE with all the detailed results in S1-S4 Tables confirmed a considerable number of norovirus-related GE outbreaks 34/61 and influenza-related RTI outbreaks 46/76 .", "The median age was 86.7 years old interquartile range: 81.1-91.0 years . Virus investigations respectively 389 samples for RTI and 143 for GE with all the detailed results in S1-S4 Tables confirmed a considerable number of norovirus-related GE outbreaks 34/61 and influenza-related RTI outbreaks 46/76 . For GE outbreaks, 2524 residents were in a NoV+ context versus 1785 in a NoV-context, and for RTI outbreaks, 3479 residents were in a Flu+ context versus 2383 in a Flu-context. For GE surveillance in the NoV+ context, there were 1093 EI residents versus 1431 ENI residents, whereas in the NoV-context, there were 583 EI residents versus 1202 ENI residents. Therefore, the infection rate was higher in the NoV+ context 43.3% than in the NoV-context 32.7%, odds ratio OR : 0.63, 95% confidence interval CI : 0.56-0.72 , p < 0.001 . For RTI surveillance, the rates of infection were similar with and without confirmed influenza: 31.5% N = 1095 EI residents /3479 exposed residents vs. 30.5% N = 728 EI residents/ 2383 exposed residents, OR: 0.96, CI: 0.85-1.07, p = 0.47 .", "Therefore, the infection rate was higher in the NoV+ context 43.3% than in the NoV-context 32.7%, odds ratio OR : 0.63, 95% confidence interval CI : 0.56-0.72 , p < 0.001 . For RTI surveillance, the rates of infection were similar with and without confirmed influenza: 31.5% N = 1095 EI residents /3479 exposed residents vs. 30.5% N = 728 EI residents/ 2383 exposed residents, OR: 0.96, CI: 0.85-1.07, p = 0.47 . Moreover, infection rate in the NoV + context was higher than the three other contexts: NoV- OR: 0.63, CI: 0.56-0.72 , Flu+ OR: 0.60, CI:0.54-0.67 and Flu- OR: 0.58, CI: 0.51-0.65 . In univariate analysis, certain individual characteristics were associated with significant variations in the infection rate S5 Table . The infection rate increased with age except in the Flucontext and, decreased with LOS during GE outbreaks. The covariate adjusted analysis revealed specific significant effect modification according to sex NoV+ and LOS NoV- S6 Table .", "The infection rate increased with age except in the Flucontext and, decreased with LOS during GE outbreaks. The covariate adjusted analysis revealed specific significant effect modification according to sex NoV+ and LOS NoV- S6 Table . In analyses stratified according to virus and sex, age adjusted for LOS remained significant for Flu+ and NoV+ outbreaks males . In NoV-context, the effect modification of LOS remained significant Table 1 . Finally, when autonomy was included and adjusted for age virus, sex, LOS stratification , the less autonomous residents female/LOS<4 years/age<86/ GIR 1-2 were affected more severely by Flu+ outbreaks with specific effect modification according to age S7 Table . The study of lethality rates in infected residents over the 56 days after onset indicated that there were significant variations for RTI but no change for GE Table 2 .", "Finally, when autonomy was included and adjusted for age virus, sex, LOS stratification , the less autonomous residents female/LOS<4 years/age<86/ GIR 1-2 were affected more severely by Flu+ outbreaks with specific effect modification according to age S7 Table . The study of lethality rates in infected residents over the 56 days after onset indicated that there were significant variations for RTI but no change for GE Table 2 . Significant differences appeared after 28 days in the context of Flu+ outbreaks and other RTI outbreaks. The analysis of successive or simultaneous clusters in the same institutions was performed when the time period between the onset of symptoms of the last case in outbreak N and the onset of symptoms of the first case in outbreak N+1 was �56 days S8 Table . 44 of the 137 outbreaks 32.12% were identified, and 194 of the 3499 exposed and infected residents contracted multiple infections. The percentage of exposed and infected residents implicated in more than one virus stratification was 11.09% 194 � 2 /3499 .", "44 of the 137 outbreaks 32.12% were identified, and 194 of the 3499 exposed and infected residents contracted multiple infections. The percentage of exposed and infected residents implicated in more than one virus stratification was 11.09% 194 � 2 /3499 . Moreover, two deceased residents were included in the NoV-Na and Flu lethality analyses because death occurred within 56 days for both infections. The analysis of virus stratification of the 44 outbreaks showed the absence of successive clusters for the same category. The same analysis for the first four 7-day intervals Days 1 to 28 showed the respective values: 26 outbreaks 18.98% , 117 residents 6.69% 117 � 2 /3499 , one dead resident. Finally, according to the higher lethality impact during the first four 7-day intervals and to limit the impact of successive clusters in the same site, all cause lethality rates were studied according to individual parameters for the four 7 days intervals with the respective number of According to the surveillance type GE or RTI , the lethality rates differed significantly: 1.6% versus 3.4% respectively NoV+ and NoV-contexts, OR: 2.24, CI: 1.16-4.39, p = 0.02 and 8.3% versus 5.6% respectively Flu+ and Flu-, OR: 0.67, CI: 0.45-0.97, p = 0.04 .", "The same analysis for the first four 7-day intervals Days 1 to 28 showed the respective values: 26 outbreaks 18.98% , 117 residents 6.69% 117 � 2 /3499 , one dead resident. Finally, according to the higher lethality impact during the first four 7-day intervals and to limit the impact of successive clusters in the same site, all cause lethality rates were studied according to individual parameters for the four 7 days intervals with the respective number of According to the surveillance type GE or RTI , the lethality rates differed significantly: 1.6% versus 3.4% respectively NoV+ and NoV-contexts, OR: 2.24, CI: 1.16-4.39, p = 0.02 and 8.3% versus 5.6% respectively Flu+ and Flu-, OR: 0.67, CI: 0.45-0.97, p = 0.04 . In univariate analysis S9 Table , low autonomy status in the NoV+, Flu+ and Flu-contexts was most significantly associated with increased all-cause lethality, and age was associated with higher lethality in the Flu+ context. In the adjusted analysis, no significant statistical differences were identified in GE outbreaks. For RTI episodes, the adjusted analysis showed that autonomy had a significant impact when adjusted for sex, age or LOS Flu+ and Flu-NA and that age had a significant impact when adjusted for sex, autonomy or LOS Flu+ S10 Table . In Table 3 , the specific effects of age or autonomy were tested.", "For RTI episodes, the adjusted analysis showed that autonomy had a significant impact when adjusted for sex, age or LOS Flu+ and Flu-NA and that age had a significant impact when adjusted for sex, autonomy or LOS Flu+ S10 Table . In Table 3 , the specific effects of age or autonomy were tested. Significant OR age adjusted for autonomy were: Flu+/age �86 years compared with the <86 group , 1.97 1.19-3.25 . OR autonomy adjusted for age were for GIR 3-6 compared with GIR 1-2 : Flu+, 0.41 0.24-0.69 ; Flu-, 0.42 0.20, 0.90 . Finally, despite the low number of residents and deaths per category, and consequently the limited robustness of the results, autonomy adjusted for age with stratification according to virus, sex and LOS showed that the effects were higher among subgroups of less autonomous residents female or male/LOS<4 years/GIR 1-2 in Flu+ outbreaks, and there was also higher mortality in the small subgroup of autonomous men with LOS � 4 years higher mortality S11 Table . In the Flu+ context, data regarding vaccination status and oseltamivir prescriptions were available but not used in this study.", "Finally, despite the low number of residents and deaths per category, and consequently the limited robustness of the results, autonomy adjusted for age with stratification according to virus, sex and LOS showed that the effects were higher among subgroups of less autonomous residents female or male/LOS<4 years/GIR 1-2 in Flu+ outbreaks, and there was also higher mortality in the small subgroup of autonomous men with LOS � 4 years higher mortality S11 Table . In the Flu+ context, data regarding vaccination status and oseltamivir prescriptions were available but not used in this study. In the present study, surveillance data obtained during GE and RTI outbreaks in nursing homes were used to construct stratified analyses and to identify specific infection and all-cause lethality rates according to the residents' individual characteristics. The infection rates observed here were similar to those found in previous studies of NoV and Influenza outbreaks odds of being infected during a Flu+ outbreak were around 40% less than during a NoV+ outbreak . Reported infection rates were close to 30.0% in influenza outbreaks and 40.0% in NoV outbreaks . Older age appeared to increase the likelihood of GE and influenza infection, with increasing rates among older residents.", "Reported infection rates were close to 30.0% in influenza outbreaks and 40.0% in NoV outbreaks . Older age appeared to increase the likelihood of GE and influenza infection, with increasing rates among older residents. Age is a well-known factor for influenza and norovirus severity in the elderly and in nursing homes . For NoV, the highest incidence estimates 5-year age strata was found in the �85 year-category approximately 800 men and for 1,400 women per 100,000 inhabitants . In our study, univariate analysis NoV+ showed that the odds of being infected were 1.5 to 1.6 times higher if a resident was older than 85. Moreover, an adjusted analysis of GE outbreaks highlighted different effects among subgroups of residents according to sex and LOS.", "In our study, univariate analysis NoV+ showed that the odds of being infected were 1.5 to 1.6 times higher if a resident was older than 85. Moreover, an adjusted analysis of GE outbreaks highlighted different effects among subgroups of residents according to sex and LOS. Indeed, multiple and/or repeated exposure to GE viruses while institutionalized may lead to susceptibility or possible increased immunity in some residents . For the sex variable, two factors could explain the effect: a possible selection bias with men reporting mild infections less than women particularly in the <86 years subgroup or that male susceptibility was different age, LOS, immunity,. . . .", ". . . A German study from 2013 also reported a greater impact in women . Moreover, when age analysis was stratified by sex and LOS, no Epidemic impacts in nursing homes significant impact was observed in women; the only significant differences were fewer infections in men in the <86-subgroup except in the NoV-with LOS <4 years . For the RTI outbreaks, sex and LOS variables did not have a significant effect. In residents older than 86, the odds of being infected in Flu+ context were 1.5 times higher for women and 1.7 for men.", "For the RTI outbreaks, sex and LOS variables did not have a significant effect. In residents older than 86, the odds of being infected in Flu+ context were 1.5 times higher for women and 1.7 for men. In univariate analysis, contrary to the other virus contexts where odds ratios were rarely above 2, residents over 95 years old had increased odds of infection of � 2.8 compared with the 70-year-old category, and for the 100 year-old group the odds were approximately 3.8. In the Flu+ context, autonomy adjusted for age virus, sex and LOS stratification revealed a possible increase in infection rates among less autonomous residents. A previous study found that when elderly residents were exposed to the A H3N2 virus, there were higher rates of infection and reinfection, and more significant effects on the institution than with other influenza types/subtypes. In the community, the relative illness ratio RIR in the .", "A previous study found that when elderly residents were exposed to the A H3N2 virus, there were higher rates of infection and reinfection, and more significant effects on the institution than with other influenza types/subtypes. In the community, the relative illness ratio RIR in the . The incidence of influenza infection and the associated risks were well described by age group, but the specific impact according to age was not studied. The results of this work highlighted the specific age distribution of influenza illnesses among the nursing home residents and the more significant impact among the older residents. This specific susceptibility could be a critical factor in the institutional exposure and dissemination of influenza and could partly explain the high infection impact in the elderly institutional population. In this work, autonomy status was not the main factor associated with infection no significant impact in GE and in Flu-contexts .", "This specific susceptibility could be a critical factor in the institutional exposure and dissemination of influenza and could partly explain the high infection impact in the elderly institutional population. In this work, autonomy status was not the main factor associated with infection no significant impact in GE and in Flu-contexts . However, in Flu+ outbreaks, a high level of dependency was associated with a higher risk of falling ill. This observation implies that staff could play a role in the spread of infection highly dependent and less mobile residents are less likely to contaminate themselves or that the more active residents may be less fragile and/or have a greater involvement in the recommended infection control measures. Finally, improving compliance with personal hygiene measures both for nursing staff and residents might be expected to have a beneficial effect on infection rates. Previous studies identified higher NoV infection rates in highly dependent individuals, but the results were not adjusted for age and LOS to take into account the potential correlation with the autonomy status .", "Finally, improving compliance with personal hygiene measures both for nursing staff and residents might be expected to have a beneficial effect on infection rates. Previous studies identified higher NoV infection rates in highly dependent individuals, but the results were not adjusted for age and LOS to take into account the potential correlation with the autonomy status . Lethality is difficult to assess in nursing homes because death is frequent. Our GE and RTI episodes occurred during the winter seasons, and there are possible interactions between outbreaks and increased mortality at this time of the year . The all-cause lethality rate of the infected residents in our study reflected global mortality including GE and RTI outbreaks and the global epidemiological context. Not surprisingly, a higher all-cause lethality rate was observed in the influenza contexts, as reported in previous studies .", "The all-cause lethality rate of the infected residents in our study reflected global mortality including GE and RTI outbreaks and the global epidemiological context. Not surprisingly, a higher all-cause lethality rate was observed in the influenza contexts, as reported in previous studies . Age and autonomy had similar effects in the different contexts, but in GE and to a lesser degree in Flu-outbreaks, the relatively small number of deaths could have limited the power of the statistical tests. In nursing homes, residents are generally discharged due to death. The number of residents lost to follow up was low 0.14% in the first 28 days , so the 7-day interval turnover rate calculated on the base of the median LOS provides a good indication of the average case fatality rate. The lethality rate for NoV+ outbreaks was similar to the estimated 7-day interval turnover rate 1.6% indicating that this context had a limited impact on the death rate.", "The number of residents lost to follow up was low 0.14% in the first 28 days , so the 7-day interval turnover rate calculated on the base of the median LOS provides a good indication of the average case fatality rate. The lethality rate for NoV+ outbreaks was similar to the estimated 7-day interval turnover rate 1.6% indicating that this context had a limited impact on the death rate. The all-cause lethality rate was most affected by age and autonomy. Both individual characteristics were significant in the Flu+ outbreaks, and autonomy adjusted for age was significant in the Flu-episodes. The influence of age on mortality in a context of influenza has already been described: a very high mortality rate 831/100,000 inhabitants was reported in persons 90 years of age and older compared with those aged 65-69 years 23/100,000 inhabitants . In our univariate analysis, the higher risk was observed in the �90 group whose risk of death was at least 2.6 higher than the <70 group.", "The influence of age on mortality in a context of influenza has already been described: a very high mortality rate 831/100,000 inhabitants was reported in persons 90 years of age and older compared with those aged 65-69 years 23/100,000 inhabitants . In our univariate analysis, the higher risk was observed in the �90 group whose risk of death was at least 2.6 higher than the <70 group. When adjusted for autonomy, the impact of age was not significant in more autonomous residents in the Flu+ context and not at all in the Flucontext. The opposite analysis autonomy adjusted for age showed higher global impact in the less autonomous group Flu- or only in the �86 age group Flu+ . Age and autonomy are a reflection of resident's level of frailty. Clinical frailty scores were not used in this study, but in a previous study of patients with critical illness, they were associated with greater mortality, regardless of age .", "Age and autonomy are a reflection of resident's level of frailty. Clinical frailty scores were not used in this study, but in a previous study of patients with critical illness, they were associated with greater mortality, regardless of age . This suggests that in addition to age, autonomy can be a valuable indicator for the assessment of outbreak impact in outbreak surveillance. Other studies have suggested that age and certain comorbidities are independent risk factors for the influenza mortality rate or that mortality increase according to the number of risk factors . Comorbidities and underlying diseases of various severities could reflect overall frailty and consequently the risk of death. In nursing homes, information about autonomy and age are easier to collect and interpret than data on comorbidities.", "Comorbidities and underlying diseases of various severities could reflect overall frailty and consequently the risk of death. In nursing homes, information about autonomy and age are easier to collect and interpret than data on comorbidities. These various approaches should be evaluated and compared in the goal of optimizing risk assessment among nursing home residents. The present work has two main limitations. First, the virus information was incomplete limited identification, mainly influenza rapid tests for the RTI . Consequently, some episodes in the levels with no available identification may also have been associated with influenza or norovirus, and multiple contaminations could have been underestimated or not taken into account.", "First, the virus information was incomplete limited identification, mainly influenza rapid tests for the RTI . Consequently, some episodes in the levels with no available identification may also have been associated with influenza or norovirus, and multiple contaminations could have been underestimated or not taken into account. Moreover, vaccination and oseltamivir prescriptions were recorded but not included because the influenza genotype was not determined and identification was limited. Secondly, the deaths of uninfected residents were not recorded in this protocol even though such data would have provided valuable information about the global epidemiological context. In conclusion, specific susceptibility patterns were observed among exposed residents. In this cohort of nursing homes, infection rates varied according to virus, sex, length of stay and age, and there were major differences in lethality depending on virus, age and autonomy score.", "In conclusion, specific susceptibility patterns were observed among exposed residents. In this cohort of nursing homes, infection rates varied according to virus, sex, length of stay and age, and there were major differences in lethality depending on virus, age and autonomy score. The collected data were easy to record and could be used to improve the characterization of seasonal outbreaks in nursing homes, whose residents are particularly vulnerable. Finally, as the average age and dependency level of residents continues to increase, subsequently increasing the risk of infection and death, health care staff will have to be increasingly vigilant during seasonal outbreaks and targeted interventions should be implemented. Supporting information S1 Data. CSV S1 R Codes.", "Supporting information S1 Data. CSV S1 R Codes. R S1 Table. XLSX S10 Table. XLSX S11 Table. XLSX" ]

[ "BACKGROUND: Gastroenteritis GE and respiratory tract infection RTI outbreaks are a significant issue in nursing homes. This study aimed to describe GE and RTI outbreaks with infection and all-cause lethality rates according to the individual characteristics of nursing home residents. METHODS: Clinical and virological surveillance were conducted 2007 to 2018 . Virus stratifications for the analysis were: outbreaks with positive norovirus or influenza identifications respectively NoV+ or Flu+ , episodes with no NoV or influenza identification or testing respectively NoV- or Flu- . Associations between individual variables sex, age, length of stay LOS , autonomy status and infection and lethality rates were tested with univariate and Mantel-Haenszel MH methods. RESULTS: 61 GE outbreaks and 76 RTI oubreaks total 137 outbreaks were recorded involving respectively 4309 and 5862 residents.", "Associations between individual variables sex, age, length of stay LOS , autonomy status and infection and lethality rates were tested with univariate and Mantel-Haenszel MH methods. RESULTS: 61 GE outbreaks and 76 RTI oubreaks total 137 outbreaks were recorded involving respectively 4309 and 5862 residents. In univariate analysis, higher infection rates and age were associated in NoV+, NoV-, and Flu+ contexts, and lower infection rates were associated with longer stays NoV+ and NoV- . In MH stratified analysis virus, sex female/male adjusted for LOS <4 or ≥4 years , the odds of being infected remained significant among older residents ≥86 years : NoV+/male Odds ratio OR MH : 1.64, 95% confidence interval CI : 1.16–2.30 and Flu+/female and male respectively OR MH : 1.50, CI: 1.27–1.79 and 1.73, CI: 1.28–2.33 . In univariate analysis, lower autonomy status NoV+, Flu+ and Flu- and increased age Flu+ were associated with higher lethality. In MH adjusted analysis, significant OR age adjusted for autonomy was: Flu+/ ≥86 years compared with <86 years, 1.97 1.19–3.25 and OR autonomy adjusted for age for the more autonomous group compared with the less autonomous group was: Flu+, 0.41 0.24–0.69 ; Flu-, 0.42 0.20, 0.90 .", "In univariate analysis, lower autonomy status NoV+, Flu+ and Flu- and increased age Flu+ were associated with higher lethality. In MH adjusted analysis, significant OR age adjusted for autonomy was: Flu+/ ≥86 years compared with <86 years, 1.97 1.19–3.25 and OR autonomy adjusted for age for the more autonomous group compared with the less autonomous group was: Flu+, 0.41 0.24–0.69 ; Flu-, 0.42 0.20, 0.90 . CONCLUSION: The residents of nursing homes are increasingly elderly and dependent. The specific infection and lethality risks according to these two factors indicate that surveillance and infection control measures are essential and of high priority. Text: Introduction Gastroenteritis GE and respiratory tract infection RTI outbreaks represent a significant burden of illness in nursing homes. Viruses cause the majority of these outbreaks, and noroviruses and influenza viruses are the most common pathogens .", "Text: Introduction Gastroenteritis GE and respiratory tract infection RTI outbreaks represent a significant burden of illness in nursing homes. Viruses cause the majority of these outbreaks, and noroviruses and influenza viruses are the most common pathogens . Previous studies have suggested that viral respiratory infections and norovirus outbreaks are a common cause of hospitalization or death, particularly among elderly individuals . The impact of outbreaks has been described in terms of both frequency and epidemiology, but little is known about infection rates and all-cause lethality in GE and RTI nursing home outbreaks in relation to the individual characteristics of the residents . The residents of these institutions are increasingly elderly and dependent, and the impact of this trend on the seasonal outbreak burden requires in-depth investigation. The results of studies focused on this issue could yield valuable information for nursing homes, allowing them to adapt their infection control strategies, in particular for improved assessment of infection risk.", "The residents of these institutions are increasingly elderly and dependent, and the impact of this trend on the seasonal outbreak burden requires in-depth investigation. The results of studies focused on this issue could yield valuable information for nursing homes, allowing them to adapt their infection control strategies, in particular for improved assessment of infection risk. Our objective was to describe GE and RTI infection and all-cause lethality rates according to the individual characteristics of nursing home residents sex, age, length of stay, autonomy status , and to identify specific susceptibility patterns related to these types of viral outbreaks in these facilities. The present study explored outbreaks in 14 sites 28 units with geriatric nursing home activities for a total of 1121 beds caring for dependent people in southern Alsace an area in northeastern France . Data were collected between September 2007 and August 2018 . Each site was geographically independent and autonomous for social and care management.", "Data were collected between September 2007 and August 2018 . Each site was geographically independent and autonomous for social and care management. Units were located within the larger sites and were defined as a place having a dedicated team at one location. During outbreaks at one site, only the residents in the units with confirmed cases were included. Outbreak inclusion depended on institutional alert to the hygiene team. Surveillance was done in each unit independently, and the members of staff had to inform a physician or charge nurse when two or more potential related cases of pneumonia or GE were observed within four days and when three or more cases were observed for other RTI.", "Outbreak inclusion depended on institutional alert to the hygiene team. Surveillance was done in each unit independently, and the members of staff had to inform a physician or charge nurse when two or more potential related cases of pneumonia or GE were observed within four days and when three or more cases were observed for other RTI. Units also had to inform the hygiene team when these threshold values were exceeded. For influenza, the first suspected case led to a local alert and the hygiene team was contacted. A practitioner from the hygiene team collected the information and evaluated the clinical signs, the virology information and the epidemiological context with the physician in the affected unit. The detected cluster was only put under surveillance if the hygiene team considered that there was a potential outbreak phenomenon.", "A practitioner from the hygiene team collected the information and evaluated the clinical signs, the virology information and the epidemiological context with the physician in the affected unit. The detected cluster was only put under surveillance if the hygiene team considered that there was a potential outbreak phenomenon. The duration of 4 days was in relation with the national protocol with alert to the authorities when 5 cases occurred within 4 days . On a local level and in addition to the clusters reported to the authorities, clusters with at least 3 cases within a period of seven days in one unit could be recorded if they were reported to the hygiene team. Because several outbreaks could potentially occur in the same unit during the surveillance period, a resident could be included repeatedly in different clusters. As a result, the observed patterns reflected the characteristics of an institutional population with longitudinal and pluriannual exposures.", "Because several outbreaks could potentially occur in the same unit during the surveillance period, a resident could be included repeatedly in different clusters. As a result, the observed patterns reflected the characteristics of an institutional population with longitudinal and pluriannual exposures. Personal information and clinical information was collected by a practitioner from the hygiene team directly from the residents' health care records. Personal information was collected for all those present the first day of the outbreak. The collected information included: month and year of birth, sex, date of arrival at the nursing home and autonomy status. The autonomy status of residents in French nursing homes is assessed using the AGGIR scale Autonomy Gerontology Groups Iso-Resources , which is the legal instrument for evaluating dependency in the elderly and whose primary purpose is the allocation of means and resources .", "The collected information included: month and year of birth, sex, date of arrival at the nursing home and autonomy status. The autonomy status of residents in French nursing homes is assessed using the AGGIR scale Autonomy Gerontology Groups Iso-Resources , which is the legal instrument for evaluating dependency in the elderly and whose primary purpose is the allocation of means and resources . With the AGGIR scale, autonomy is classified into 6 Iso-Resource Groups GIR : GIR 1 bedridden or armchair-bound persons, mental functions seriously altered and requiring continuous presence , GIR 2 bedridden or armchair-bound persons, mental functions not totally altered and requiring assistance in most activities of daily living, or mental functions altered with preserved ability to get around , GIR 3 preserved mental autonomy with partially preserved motor autonomy and assistance several times a day for physical autonomy , GIR 4 moves around the home and sometimes assistance for washing, dressing, physical activities or eating , GIR 5 only occasional assistance for washing, meal preparation, and housework , GIR 6 autonomy for essential tasks of daily living . In outbreaks where the influenza virus was identified, influenza vaccination status and oseltamivir prescriptions were recorded as well. A file is transmitted in the Supporting Information with all previous data S1 Data . GE was defined as the sudden onset of vomiting and/or diarrhea over a 24 h period: i diarrhea �3 episodes, ii and/or vomiting �3 episodes, iii or diarrhea or vomiting <3 episodes with two or more other symptoms diarrhea, vomiting, stomach ache, abdominal cramps, nausea, fever, mucus in stools .", "A file is transmitted in the Supporting Information with all previous data S1 Data . GE was defined as the sudden onset of vomiting and/or diarrhea over a 24 h period: i diarrhea �3 episodes, ii and/or vomiting �3 episodes, iii or diarrhea or vomiting <3 episodes with two or more other symptoms diarrhea, vomiting, stomach ache, abdominal cramps, nausea, fever, mucus in stools . RTI presentation in older adults may be atypical, like for other acute illnesses in this age group . We used the recommended definitions for RTI surveillance in geriatric units, divided in 3 subcategories: i common cold syndromes or pharyngitis at least two of the following criteria: runny nose or sneezing, stuffy nose i.e. congestion , sore throat or hoarseness or difficulty swallowing, dry cough, swollen or tender glands in the neck cervical lymphadenopathy , ii influenza-like illness both the following criteria must be met: fever AND at least three other symptoms chills, new headache or eye pain, myalgia or body aches, malaise or loss of appetite, sore throat, new or increased dry cough and iii lower respiratory tract infection both of the following criteria must be met: at least two respiratory signs or symptoms new or increased cough, new/increased sputum production, O 2 saturation <94% or reduced >3% from baseline, abnormal lung examination new or changed , pleuritic chest pain, respiratory rate �25 breaths/min AND one or more constitutional signs/symptoms fever, leukocytosis, confusion, acute functional decline . Infection corresponding to one of these three subcategories was included in this study and classified as RTI.", "congestion , sore throat or hoarseness or difficulty swallowing, dry cough, swollen or tender glands in the neck cervical lymphadenopathy , ii influenza-like illness both the following criteria must be met: fever AND at least three other symptoms chills, new headache or eye pain, myalgia or body aches, malaise or loss of appetite, sore throat, new or increased dry cough and iii lower respiratory tract infection both of the following criteria must be met: at least two respiratory signs or symptoms new or increased cough, new/increased sputum production, O 2 saturation <94% or reduced >3% from baseline, abnormal lung examination new or changed , pleuritic chest pain, respiratory rate �25 breaths/min AND one or more constitutional signs/symptoms fever, leukocytosis, confusion, acute functional decline . Infection corresponding to one of these three subcategories was included in this study and classified as RTI. For both infection types, the practitioner from the hygiene team obtained clinical information from the patient's health care records, and members of the health care team were consulted if necessary to complete any missing information. At the end of the episode within seven days after the last identified case , case inclusion as exposed and not infected ENI or exposed and infected EI was determined with a resident physician. In order to study the lethality, the presence of each infected resident was evaluated once at least 56 days after the last case of each outbreak. Each resident was followed up retrospectively during eighth 7-day interval I n, n = 1 to 8, total 56 days, between the date of onset of symptoms and the fifty-sixth day of the studied period with three different possibilities: present alive and officially residing in the institution , lost to follow-up alive at the date of departure but no longer residing in the institution return home, transfer to another institution or death death recorded in the health care record .", "In order to study the lethality, the presence of each infected resident was evaluated once at least 56 days after the last case of each outbreak. Each resident was followed up retrospectively during eighth 7-day interval I n, n = 1 to 8, total 56 days, between the date of onset of symptoms and the fifty-sixth day of the studied period with three different possibilities: present alive and officially residing in the institution , lost to follow-up alive at the date of departure but no longer residing in the institution return home, transfer to another institution or death death recorded in the health care record . The dates of death and lost to follow-up were recorded. Testing for the virus was not systematic and was decided by the physicians in each institution in the presence of clinical signs. For GE, stool samples were sent to the National Reference Centre for Gastroenteritis Viruses in Dijon for laboratory testing, as previously described . For RTI surveillance, rapid tests were used to identify the influenza virus.", "For GE, stool samples were sent to the National Reference Centre for Gastroenteritis Viruses in Dijon for laboratory testing, as previously described . For RTI surveillance, rapid tests were used to identify the influenza virus. The rapid immunoassay diagnosis tests used for influenza detection were: Clearview1 Exact Influenza A and B Inverness Medical, Cologne, Germany from 2007 to 2014 and InfluenzaTop1 Alldiag, Strasbourg, France from 2014 to 2018. Given the low sensitivity of influenza rapid tests, they were no longer used once the control measures had been implemented and the influenza outbreak was under control. Samples were also occasionally sent to hospital laboratories or to the National Reference Centre for Influenza Viruses to detect viruses with real-time RT-PCR . Most testing targeted the norovirus NoV and influenza virus, but other tests were occasionally performed by the National Reference Centre for Influenza Viruses rhinovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza 1, 2, 3 and 4, and coronavirus and the National Reference Centre for Enteric Viruses rotavirus, astrovirus, and adenovirus .", "Samples were also occasionally sent to hospital laboratories or to the National Reference Centre for Influenza Viruses to detect viruses with real-time RT-PCR . Most testing targeted the norovirus NoV and influenza virus, but other tests were occasionally performed by the National Reference Centre for Influenza Viruses rhinovirus, respiratory syncytial virus, human metapneumovirus, parainfluenza 1, 2, 3 and 4, and coronavirus and the National Reference Centre for Enteric Viruses rotavirus, astrovirus, and adenovirus . Because testing for the viruses was variable from one institution/physician to another, not used in some outbreaks, types of virus sought and considering the poor sensitivity of the rapid influenza tests, these two sources of data were used to define the epidemiological context of confirmed outbreaks. Consequently, individual cases were included consistently in all episodes according to clinical signs and medical evaluation. When virus testing was negative, the clinical signs were recorded and medical evaluation was used as previously to classify the included residents as infected or not infected. The epidemiological context of each outbreak was defined according to whether the virus had been identified or not.", "When virus testing was negative, the clinical signs were recorded and medical evaluation was used as previously to classify the included residents as infected or not infected. The epidemiological context of each outbreak was defined according to whether the virus had been identified or not. One or more positive samples led to the qualification of a NoV NoV+ or flu Flu+ context. The other episodes were qualified as flu or NoV outbreaks with no specific identification or testing NoV-and Flu- . Flu and NoV contexts did not eliminate other potential enteric or respiratory pathogens. Sex, age, length of stay LOS, in years and autonomy status were described for all exposed residents.", "Flu and NoV contexts did not eliminate other potential enteric or respiratory pathogens. Sex, age, length of stay LOS, in years and autonomy status were described for all exposed residents. Influenza vaccination and oseltamivir administration rates were calculated for confirmed influenza outbreaks. Dichotomous or categorical variables were expressed as percentages. In univariate analysis, the categories were specific in order to obtain a precise description of the age and LOS variables. Class intervals were 5 years for age and one year for LOS. The residents classified as GIR 4 to 6 sometimes, occasional and no assistance were grouped together because they were few.", "Class intervals were 5 years for age and one year for LOS. The residents classified as GIR 4 to 6 sometimes, occasional and no assistance were grouped together because they were few. For the multi-level analysis with 2x2 tables, a median value was used to define the two-level age categories. For LOS, assessing the longest stays was necessary to identify the effect of longer exposure in a nursing home. Consequently, a four-year cutoff was chosen to create the two categories. For autonomy, the two most dependent categories GIR � 2 were grouped together and compared with the more autonomous categories GIR � 3 .", "Consequently, a four-year cutoff was chosen to create the two categories. For autonomy, the two most dependent categories GIR � 2 were grouped together and compared with the more autonomous categories GIR � 3 . The outbreak epidemiological contexts were used with the four categories: NoV+, Flu+, NoV-and flu-. Other GE or RTI viruses were occasionally identified, but the number of results was too limited to develop separate analyses. However, all the results are available in the tables about the virus investigations along with NoV and influenza identifications. For the different categories, infection rate EI/Exposed Residents ER , in percentage was calculated according to sex, age group, LOS and autonomy status.", "However, all the results are available in the tables about the virus investigations along with NoV and influenza identifications. For the different categories, infection rate EI/Exposed Residents ER , in percentage was calculated according to sex, age group, LOS and autonomy status. To investigate all-cause lethality and define the appropriate period for the 56-day monitoring D 1 to 56 , the all-cause lethality rate per 7-day interval LR n /I n, n = 1 to 8 was calculated: number of deaths during interval I n / EI alive the first day of n th studied interval minus lost to follow-up EI during the interval I n � 100 . Seeing as successive clusters could occur within the same site, potentially influencing allcause lethality, the serial interval in days SI d was calculated. The SI d was the time period between the onset of symptoms of the last case in initial outbreak N and the onset of symptoms of the first case in the following outbreak N+1 . Investigations were performed when SI d was shorter or equal to the length of the previous D 1 to 56 and the following parameters were evaluated for these specific situations: number of episodes, residents infected in both outbreaks, and death among the identified individuals.", "The SI d was the time period between the onset of symptoms of the last case in initial outbreak N and the onset of symptoms of the first case in the following outbreak N+1 . Investigations were performed when SI d was shorter or equal to the length of the previous D 1 to 56 and the following parameters were evaluated for these specific situations: number of episodes, residents infected in both outbreaks, and death among the identified individuals. Finally, according to the death rate and the impact of successive outbreaks, the number of 7-day intervals N.I n to take into account was defined, and the all-cause lethality rate was analyzed during these periods I 1 to N th .I n or D 1 to 7 � N . All-cause lethality rates were calculated with the following formula: number of deaths from D 1 to 7 � N / EI number at D 1 minus lost to follow-up among EI during the period D 1 to 7 � N � 100 . Estimation of the turnover rate per 7-day interval among the infected residents was calculated on the base of the LOS median in years with the following formula: proportion of discharged residents: 50.0% in the case of the median / median LOS � 365 /7 . The average rate of residents discharged per 7-day period was calculated: number of lost to follow up during the period D 1 to 7 � N /number of exposed and infected residents at D 1 /N 7-day interval � 100.", "Estimation of the turnover rate per 7-day interval among the infected residents was calculated on the base of the LOS median in years with the following formula: proportion of discharged residents: 50.0% in the case of the median / median LOS � 365 /7 . The average rate of residents discharged per 7-day period was calculated: number of lost to follow up during the period D 1 to 7 � N /number of exposed and infected residents at D 1 /N 7-day interval � 100. As some residents were included in several outbreaks during the surveillance, the observations were not completely independent; non-parametric tests were used as a result. In univariate analysis, Chi-square or Fisher exact tests expected number of frequencies fewer than 5 were used to compare infection and lethality rates according to the studied parameters and the odds ratio was calculated by median-unbiased estimation. The Kruskal-Wallis test was used to compare median values. Confidence intervals for medians were calculated with bootstrap methods.", "The Kruskal-Wallis test was used to compare median values. Confidence intervals for medians were calculated with bootstrap methods. Covariate adjusted analyses were performed with two tables 2x2 . The respective impact of each individual factor was tested with Mantel-Haenszel chi-squared tests. The equality of the stratum odds ratios was tested with the Woolf test of homogeneity. Finally, for each virus context, multiple tables 2x2 were generated and tested with confounding variables, effect modifiers or covariables. Statistical analyses were done using R for Mas OS X version R 3.4.1 software with RStudio version 1.0.153. A file is transmitted in the Supporting Information with all R codes and the packages used S1 R Codes .", "Statistical analyses were done using R for Mas OS X version R 3.4.1 software with RStudio version 1.0.153. A file is transmitted in the Supporting Information with all R codes and the packages used S1 R Codes . Differences were considered significant at p � 0.05. The French Data Protection Authority approved data collection and analysis DE-2013-074 and the local ethics committee Espace Local de Réflexion Ethique, Centre Hospitalier de Rouffach approved the study protocol ERLE-32 . According to the French law for biomedical research and human experimentation, individual written consent was not required from the patients or their relatives for data collection. Each year, the referring local practitioner of the study coordinated with the doctors working in the nursing home.", "According to the French law for biomedical research and human experimentation, individual written consent was not required from the patients or their relatives for data collection. Each year, the referring local practitioner of the study coordinated with the doctors working in the nursing home. At the beginning of the surveillance period, information regarding participation in the study was displayed in the family vising area, including a document about their right to access and rectify personal data. After collection, data were rendered anonymous. No specific authorization was needed to retrospectively analyze anonymous data collected during routine care in the context of routine surveillance. A total of 137 outbreaks were recorded in the 14 sites.", "No specific authorization was needed to retrospectively analyze anonymous data collected during routine care in the context of routine surveillance. A total of 137 outbreaks were recorded in the 14 sites. RTI outbreaks were more frequent than GE outbreaks 76 outbreaks and 5862 exposed residents vs. 61 outbreaks and 4309 exposed residents, respectively . Overall, 7643 of the exposed residents were women and 2528 were men. The median age was 86.7 years old interquartile range: 81.1-91.0 years . Virus investigations respectively 389 samples for RTI and 143 for GE with all the detailed results in S1-S4 Tables confirmed a considerable number of norovirus-related GE outbreaks 34/61 and influenza-related RTI outbreaks 46/76 .", "The median age was 86.7 years old interquartile range: 81.1-91.0 years . Virus investigations respectively 389 samples for RTI and 143 for GE with all the detailed results in S1-S4 Tables confirmed a considerable number of norovirus-related GE outbreaks 34/61 and influenza-related RTI outbreaks 46/76 . For GE outbreaks, 2524 residents were in a NoV+ context versus 1785 in a NoV-context, and for RTI outbreaks, 3479 residents were in a Flu+ context versus 2383 in a Flu-context. For GE surveillance in the NoV+ context, there were 1093 EI residents versus 1431 ENI residents, whereas in the NoV-context, there were 583 EI residents versus 1202 ENI residents. Therefore, the infection rate was higher in the NoV+ context 43.3% than in the NoV-context 32.7%, odds ratio OR : 0.63, 95% confidence interval CI : 0.56-0.72 , p < 0.001 . For RTI surveillance, the rates of infection were similar with and without confirmed influenza: 31.5% N = 1095 EI residents /3479 exposed residents vs. 30.5% N = 728 EI residents/ 2383 exposed residents, OR: 0.96, CI: 0.85-1.07, p = 0.47 .", "Therefore, the infection rate was higher in the NoV+ context 43.3% than in the NoV-context 32.7%, odds ratio OR : 0.63, 95% confidence interval CI : 0.56-0.72 , p < 0.001 . For RTI surveillance, the rates of infection were similar with and without confirmed influenza: 31.5% N = 1095 EI residents /3479 exposed residents vs. 30.5% N = 728 EI residents/ 2383 exposed residents, OR: 0.96, CI: 0.85-1.07, p = 0.47 . Moreover, infection rate in the NoV + context was higher than the three other contexts: NoV- OR: 0.63, CI: 0.56-0.72 , Flu+ OR: 0.60, CI:0.54-0.67 and Flu- OR: 0.58, CI: 0.51-0.65 . In univariate analysis, certain individual characteristics were associated with significant variations in the infection rate S5 Table . The infection rate increased with age except in the Flucontext and, decreased with LOS during GE outbreaks. The covariate adjusted analysis revealed specific significant effect modification according to sex NoV+ and LOS NoV- S6 Table .", "The infection rate increased with age except in the Flucontext and, decreased with LOS during GE outbreaks. The covariate adjusted analysis revealed specific significant effect modification according to sex NoV+ and LOS NoV- S6 Table . In analyses stratified according to virus and sex, age adjusted for LOS remained significant for Flu+ and NoV+ outbreaks males . In NoV-context, the effect modification of LOS remained significant Table 1 . Finally, when autonomy was included and adjusted for age virus, sex, LOS stratification , the less autonomous residents female/LOS<4 years/age<86/ GIR 1-2 were affected more severely by Flu+ outbreaks with specific effect modification according to age S7 Table . The study of lethality rates in infected residents over the 56 days after onset indicated that there were significant variations for RTI but no change for GE Table 2 .", "Finally, when autonomy was included and adjusted for age virus, sex, LOS stratification , the less autonomous residents female/LOS<4 years/age<86/ GIR 1-2 were affected more severely by Flu+ outbreaks with specific effect modification according to age S7 Table . The study of lethality rates in infected residents over the 56 days after onset indicated that there were significant variations for RTI but no change for GE Table 2 . Significant differences appeared after 28 days in the context of Flu+ outbreaks and other RTI outbreaks. The analysis of successive or simultaneous clusters in the same institutions was performed when the time period between the onset of symptoms of the last case in outbreak N and the onset of symptoms of the first case in outbreak N+1 was �56 days S8 Table . 44 of the 137 outbreaks 32.12% were identified, and 194 of the 3499 exposed and infected residents contracted multiple infections. The percentage of exposed and infected residents implicated in more than one virus stratification was 11.09% 194 � 2 /3499 .", "44 of the 137 outbreaks 32.12% were identified, and 194 of the 3499 exposed and infected residents contracted multiple infections. The percentage of exposed and infected residents implicated in more than one virus stratification was 11.09% 194 � 2 /3499 . Moreover, two deceased residents were included in the NoV-Na and Flu lethality analyses because death occurred within 56 days for both infections. The analysis of virus stratification of the 44 outbreaks showed the absence of successive clusters for the same category. The same analysis for the first four 7-day intervals Days 1 to 28 showed the respective values: 26 outbreaks 18.98% , 117 residents 6.69% 117 � 2 /3499 , one dead resident. Finally, according to the higher lethality impact during the first four 7-day intervals and to limit the impact of successive clusters in the same site, all cause lethality rates were studied according to individual parameters for the four 7 days intervals with the respective number of According to the surveillance type GE or RTI , the lethality rates differed significantly: 1.6% versus 3.4% respectively NoV+ and NoV-contexts, OR: 2.24, CI: 1.16-4.39, p = 0.02 and 8.3% versus 5.6% respectively Flu+ and Flu-, OR: 0.67, CI: 0.45-0.97, p = 0.04 .", "The same analysis for the first four 7-day intervals Days 1 to 28 showed the respective values: 26 outbreaks 18.98% , 117 residents 6.69% 117 � 2 /3499 , one dead resident. Finally, according to the higher lethality impact during the first four 7-day intervals and to limit the impact of successive clusters in the same site, all cause lethality rates were studied according to individual parameters for the four 7 days intervals with the respective number of According to the surveillance type GE or RTI , the lethality rates differed significantly: 1.6% versus 3.4% respectively NoV+ and NoV-contexts, OR: 2.24, CI: 1.16-4.39, p = 0.02 and 8.3% versus 5.6% respectively Flu+ and Flu-, OR: 0.67, CI: 0.45-0.97, p = 0.04 . In univariate analysis S9 Table , low autonomy status in the NoV+, Flu+ and Flu-contexts was most significantly associated with increased all-cause lethality, and age was associated with higher lethality in the Flu+ context. In the adjusted analysis, no significant statistical differences were identified in GE outbreaks. For RTI episodes, the adjusted analysis showed that autonomy had a significant impact when adjusted for sex, age or LOS Flu+ and Flu-NA and that age had a significant impact when adjusted for sex, autonomy or LOS Flu+ S10 Table . In Table 3 , the specific effects of age or autonomy were tested.", "For RTI episodes, the adjusted analysis showed that autonomy had a significant impact when adjusted for sex, age or LOS Flu+ and Flu-NA and that age had a significant impact when adjusted for sex, autonomy or LOS Flu+ S10 Table . In Table 3 , the specific effects of age or autonomy were tested. Significant OR age adjusted for autonomy were: Flu+/age �86 years compared with the <86 group , 1.97 1.19-3.25 . OR autonomy adjusted for age were for GIR 3-6 compared with GIR 1-2 : Flu+, 0.41 0.24-0.69 ; Flu-, 0.42 0.20, 0.90 . Finally, despite the low number of residents and deaths per category, and consequently the limited robustness of the results, autonomy adjusted for age with stratification according to virus, sex and LOS showed that the effects were higher among subgroups of less autonomous residents female or male/LOS<4 years/GIR 1-2 in Flu+ outbreaks, and there was also higher mortality in the small subgroup of autonomous men with LOS � 4 years higher mortality S11 Table . In the Flu+ context, data regarding vaccination status and oseltamivir prescriptions were available but not used in this study.", "Finally, despite the low number of residents and deaths per category, and consequently the limited robustness of the results, autonomy adjusted for age with stratification according to virus, sex and LOS showed that the effects were higher among subgroups of less autonomous residents female or male/LOS<4 years/GIR 1-2 in Flu+ outbreaks, and there was also higher mortality in the small subgroup of autonomous men with LOS � 4 years higher mortality S11 Table . In the Flu+ context, data regarding vaccination status and oseltamivir prescriptions were available but not used in this study. In the present study, surveillance data obtained during GE and RTI outbreaks in nursing homes were used to construct stratified analyses and to identify specific infection and all-cause lethality rates according to the residents' individual characteristics. The infection rates observed here were similar to those found in previous studies of NoV and Influenza outbreaks odds of being infected during a Flu+ outbreak were around 40% less than during a NoV+ outbreak . Reported infection rates were close to 30.0% in influenza outbreaks and 40.0% in NoV outbreaks . Older age appeared to increase the likelihood of GE and influenza infection, with increasing rates among older residents.", "Reported infection rates were close to 30.0% in influenza outbreaks and 40.0% in NoV outbreaks . Older age appeared to increase the likelihood of GE and influenza infection, with increasing rates among older residents. Age is a well-known factor for influenza and norovirus severity in the elderly and in nursing homes . For NoV, the highest incidence estimates 5-year age strata was found in the �85 year-category approximately 800 men and for 1,400 women per 100,000 inhabitants . In our study, univariate analysis NoV+ showed that the odds of being infected were 1.5 to 1.6 times higher if a resident was older than 85. Moreover, an adjusted analysis of GE outbreaks highlighted different effects among subgroups of residents according to sex and LOS.", "In our study, univariate analysis NoV+ showed that the odds of being infected were 1.5 to 1.6 times higher if a resident was older than 85. Moreover, an adjusted analysis of GE outbreaks highlighted different effects among subgroups of residents according to sex and LOS. Indeed, multiple and/or repeated exposure to GE viruses while institutionalized may lead to susceptibility or possible increased immunity in some residents . For the sex variable, two factors could explain the effect: a possible selection bias with men reporting mild infections less than women particularly in the <86 years subgroup or that male susceptibility was different age, LOS, immunity,. . . .", ". . . A German study from 2013 also reported a greater impact in women . Moreover, when age analysis was stratified by sex and LOS, no Epidemic impacts in nursing homes significant impact was observed in women; the only significant differences were fewer infections in men in the <86-subgroup except in the NoV-with LOS <4 years . For the RTI outbreaks, sex and LOS variables did not have a significant effect. In residents older than 86, the odds of being infected in Flu+ context were 1.5 times higher for women and 1.7 for men.", "For the RTI outbreaks, sex and LOS variables did not have a significant effect. In residents older than 86, the odds of being infected in Flu+ context were 1.5 times higher for women and 1.7 for men. In univariate analysis, contrary to the other virus contexts where odds ratios were rarely above 2, residents over 95 years old had increased odds of infection of � 2.8 compared with the 70-year-old category, and for the 100 year-old group the odds were approximately 3.8. In the Flu+ context, autonomy adjusted for age virus, sex and LOS stratification revealed a possible increase in infection rates among less autonomous residents. A previous study found that when elderly residents were exposed to the A H3N2 virus, there were higher rates of infection and reinfection, and more significant effects on the institution than with other influenza types/subtypes. In the community, the relative illness ratio RIR in the .", "A previous study found that when elderly residents were exposed to the A H3N2 virus, there were higher rates of infection and reinfection, and more significant effects on the institution than with other influenza types/subtypes. In the community, the relative illness ratio RIR in the . The incidence of influenza infection and the associated risks were well described by age group, but the specific impact according to age was not studied. The results of this work highlighted the specific age distribution of influenza illnesses among the nursing home residents and the more significant impact among the older residents. This specific susceptibility could be a critical factor in the institutional exposure and dissemination of influenza and could partly explain the high infection impact in the elderly institutional population. In this work, autonomy status was not the main factor associated with infection no significant impact in GE and in Flu-contexts .", "This specific susceptibility could be a critical factor in the institutional exposure and dissemination of influenza and could partly explain the high infection impact in the elderly institutional population. In this work, autonomy status was not the main factor associated with infection no significant impact in GE and in Flu-contexts . However, in Flu+ outbreaks, a high level of dependency was associated with a higher risk of falling ill. This observation implies that staff could play a role in the spread of infection highly dependent and less mobile residents are less likely to contaminate themselves or that the more active residents may be less fragile and/or have a greater involvement in the recommended infection control measures. Finally, improving compliance with personal hygiene measures both for nursing staff and residents might be expected to have a beneficial effect on infection rates. Previous studies identified higher NoV infection rates in highly dependent individuals, but the results were not adjusted for age and LOS to take into account the potential correlation with the autonomy status .", "Finally, improving compliance with personal hygiene measures both for nursing staff and residents might be expected to have a beneficial effect on infection rates. Previous studies identified higher NoV infection rates in highly dependent individuals, but the results were not adjusted for age and LOS to take into account the potential correlation with the autonomy status . Lethality is difficult to assess in nursing homes because death is frequent. Our GE and RTI episodes occurred during the winter seasons, and there are possible interactions between outbreaks and increased mortality at this time of the year . The all-cause lethality rate of the infected residents in our study reflected global mortality including GE and RTI outbreaks and the global epidemiological context. Not surprisingly, a higher all-cause lethality rate was observed in the influenza contexts, as reported in previous studies .", "The all-cause lethality rate of the infected residents in our study reflected global mortality including GE and RTI outbreaks and the global epidemiological context. Not surprisingly, a higher all-cause lethality rate was observed in the influenza contexts, as reported in previous studies . Age and autonomy had similar effects in the different contexts, but in GE and to a lesser degree in Flu-outbreaks, the relatively small number of deaths could have limited the power of the statistical tests. In nursing homes, residents are generally discharged due to death. The number of residents lost to follow up was low 0.14% in the first 28 days , so the 7-day interval turnover rate calculated on the base of the median LOS provides a good indication of the average case fatality rate. The lethality rate for NoV+ outbreaks was similar to the estimated 7-day interval turnover rate 1.6% indicating that this context had a limited impact on the death rate.", "The number of residents lost to follow up was low 0.14% in the first 28 days , so the 7-day interval turnover rate calculated on the base of the median LOS provides a good indication of the average case fatality rate. The lethality rate for NoV+ outbreaks was similar to the estimated 7-day interval turnover rate 1.6% indicating that this context had a limited impact on the death rate. The all-cause lethality rate was most affected by age and autonomy. Both individual characteristics were significant in the Flu+ outbreaks, and autonomy adjusted for age was significant in the Flu-episodes. The influence of age on mortality in a context of influenza has already been described: a very high mortality rate 831/100,000 inhabitants was reported in persons 90 years of age and older compared with those aged 65-69 years 23/100,000 inhabitants . In our univariate analysis, the higher risk was observed in the �90 group whose risk of death was at least 2.6 higher than the <70 group.", "The influence of age on mortality in a context of influenza has already been described: a very high mortality rate 831/100,000 inhabitants was reported in persons 90 years of age and older compared with those aged 65-69 years 23/100,000 inhabitants . In our univariate analysis, the higher risk was observed in the �90 group whose risk of death was at least 2.6 higher than the <70 group. When adjusted for autonomy, the impact of age was not significant in more autonomous residents in the Flu+ context and not at all in the Flucontext. The opposite analysis autonomy adjusted for age showed higher global impact in the less autonomous group Flu- or only in the �86 age group Flu+ . Age and autonomy are a reflection of resident's level of frailty. Clinical frailty scores were not used in this study, but in a previous study of patients with critical illness, they were associated with greater mortality, regardless of age .", "Age and autonomy are a reflection of resident's level of frailty. Clinical frailty scores were not used in this study, but in a previous study of patients with critical illness, they were associated with greater mortality, regardless of age . This suggests that in addition to age, autonomy can be a valuable indicator for the assessment of outbreak impact in outbreak surveillance. Other studies have suggested that age and certain comorbidities are independent risk factors for the influenza mortality rate or that mortality increase according to the number of risk factors . Comorbidities and underlying diseases of various severities could reflect overall frailty and consequently the risk of death. In nursing homes, information about autonomy and age are easier to collect and interpret than data on comorbidities.", "Comorbidities and underlying diseases of various severities could reflect overall frailty and consequently the risk of death. In nursing homes, information about autonomy and age are easier to collect and interpret than data on comorbidities. These various approaches should be evaluated and compared in the goal of optimizing risk assessment among nursing home residents. The present work has two main limitations. First, the virus information was incomplete limited identification, mainly influenza rapid tests for the RTI . Consequently, some episodes in the levels with no available identification may also have been associated with influenza or norovirus, and multiple contaminations could have been underestimated or not taken into account.", "First, the virus information was incomplete limited identification, mainly influenza rapid tests for the RTI . Consequently, some episodes in the levels with no available identification may also have been associated with influenza or norovirus, and multiple contaminations could have been underestimated or not taken into account. Moreover, vaccination and oseltamivir prescriptions were recorded but not included because the influenza genotype was not determined and identification was limited. Secondly, the deaths of uninfected residents were not recorded in this protocol even though such data would have provided valuable information about the global epidemiological context. In conclusion, specific susceptibility patterns were observed among exposed residents. In this cohort of nursing homes, infection rates varied according to virus, sex, length of stay and age, and there were major differences in lethality depending on virus, age and autonomy score.", "In conclusion, specific susceptibility patterns were observed among exposed residents. In this cohort of nursing homes, infection rates varied according to virus, sex, length of stay and age, and there were major differences in lethality depending on virus, age and autonomy score. The collected data were easy to record and could be used to improve the characterization of seasonal outbreaks in nursing homes, whose residents are particularly vulnerable. Finally, as the average age and dependency level of residents continues to increase, subsequently increasing the risk of infection and death, health care staff will have to be increasingly vigilant during seasonal outbreaks and targeted interventions should be implemented. Supporting information S1 Data. CSV S1 R Codes.", "Supporting information S1 Data. CSV S1 R Codes. R S1 Table. XLSX S10 Table. XLSX S11 Table. XLSX" ]

[ "nan Text: The main challenge may include . early identification of outbreak, . rapid expansion of patients, . high risk of nosocomial transmission, . unpredictability of size impacted, and . lack of backup resource. These challenges have caused severe shortage of healthcare workers, medical materials, and beds with isolation. The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage.", "The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage. There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis. Early identification of 2019-nCoV infection presents a major challenge for the frontline clinicians. Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever 98% , cough 76% , and diarrhea 3% , often more severe in older adults with pre-existing chronic comorbidities . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia .", "Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever 98% , cough 76% , and diarrhea 3% , often more severe in older adults with pre-existing chronic comorbidities . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia . The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation . In addition, asymptomatic cases and lack of diagnosis kits result in delayed or even missed diagnosis inevitable and makes many other patients, visitors, and healthcare workers exposed to the 2019-nCoV infection. Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital IDH .", "Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital IDH . The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH. Such a plan is mandatory for every hospital. Shenzhen city has established a preexisting Infectious Disease Epidemic Plan IDEP , which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital.", "Shenzhen city has established a preexisting Infectious Disease Epidemic Plan IDEP , which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital. Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds. 2019-nCoV patients should be admitted to singlebedded, negative pressure rooms in isolated units with intensive care and monitoring . Clinical engineering should have plans to reconstruct standard rooms . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution.", "Clinical engineering should have plans to reconstruct standard rooms . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution. Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors . More importantly, because the hospitals' ability to respond to the outbreak largely depends on their available ICU beds, the plan to increase ICU bed capacity needs to be determined. Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 95% CI, 1.4 to 3.9 , or as high as between 3.6 and 4.0 .", "Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 95% CI, 1.4 to 3.9 , or as high as between 3.6 and 4.0 . The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible . The higher viral load and aerosol-generating procedures, such as noninvasive ventilation, magnify the exposure and transmission risk . Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator.", "Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator. Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee CNHC . In addition, wellequipped fever clinic as triage station with trained staff knowing 2019-nCoV case definitions is established. For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens , and implementing droplet isolation to break the transmission chain in the healthcare setting . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers .", "For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens , and implementing droplet isolation to break the transmission chain in the healthcare setting . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers . The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020. Psychotherapists have also been invited to join medical teams to evaluate and deal with potential stress and depression for the safety of the healthcare workers. Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation ECMO according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged.", "Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation ECMO according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged. These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality. On the contrary, the previously reported mortality of 2019-nCoV patients in Wuhan ranged from 11 to 15% ." ]

[ "nan Text: The main challenge may include . early identification of outbreak, . rapid expansion of patients, . high risk of nosocomial transmission, . unpredictability of size impacted, and . lack of backup resource. These challenges have caused severe shortage of healthcare workers, medical materials, and beds with isolation. The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage.", "The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage. There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis. Early identification of 2019-nCoV infection presents a major challenge for the frontline clinicians. Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever 98% , cough 76% , and diarrhea 3% , often more severe in older adults with pre-existing chronic comorbidities . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia .", "Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever 98% , cough 76% , and diarrhea 3% , often more severe in older adults with pre-existing chronic comorbidities . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia . The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation . In addition, asymptomatic cases and lack of diagnosis kits result in delayed or even missed diagnosis inevitable and makes many other patients, visitors, and healthcare workers exposed to the 2019-nCoV infection. Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital IDH .", "Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital IDH . The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH. Such a plan is mandatory for every hospital. Shenzhen city has established a preexisting Infectious Disease Epidemic Plan IDEP , which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital.", "Shenzhen city has established a preexisting Infectious Disease Epidemic Plan IDEP , which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital. Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds. 2019-nCoV patients should be admitted to singlebedded, negative pressure rooms in isolated units with intensive care and monitoring . Clinical engineering should have plans to reconstruct standard rooms . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution.", "Clinical engineering should have plans to reconstruct standard rooms . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution. Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors . More importantly, because the hospitals' ability to respond to the outbreak largely depends on their available ICU beds, the plan to increase ICU bed capacity needs to be determined. Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 95% CI, 1.4 to 3.9 , or as high as between 3.6 and 4.0 .", "Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 95% CI, 1.4 to 3.9 , or as high as between 3.6 and 4.0 . The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible . The higher viral load and aerosol-generating procedures, such as noninvasive ventilation, magnify the exposure and transmission risk . Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator.", "Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator. Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee CNHC . In addition, wellequipped fever clinic as triage station with trained staff knowing 2019-nCoV case definitions is established. For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens , and implementing droplet isolation to break the transmission chain in the healthcare setting . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers .", "For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens , and implementing droplet isolation to break the transmission chain in the healthcare setting . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers . The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020. Psychotherapists have also been invited to join medical teams to evaluate and deal with potential stress and depression for the safety of the healthcare workers. Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation ECMO according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged.", "Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation ECMO according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged. These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality. On the contrary, the previously reported mortality of 2019-nCoV patients in Wuhan ranged from 11 to 15% ." ]

[ "nan Text: The main challenge may include . early identification of outbreak, . rapid expansion of patients, . high risk of nosocomial transmission, . unpredictability of size impacted, and . lack of backup resource. These challenges have caused severe shortage of healthcare workers, medical materials, and beds with isolation. The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage.", "The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage. There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis. Early identification of 2019-nCoV infection presents a major challenge for the frontline clinicians. Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever 98% , cough 76% , and diarrhea 3% , often more severe in older adults with pre-existing chronic comorbidities . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia .", "Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever 98% , cough 76% , and diarrhea 3% , often more severe in older adults with pre-existing chronic comorbidities . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia . The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation . In addition, asymptomatic cases and lack of diagnosis kits result in delayed or even missed diagnosis inevitable and makes many other patients, visitors, and healthcare workers exposed to the 2019-nCoV infection. Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital IDH .", "Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital IDH . The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH. Such a plan is mandatory for every hospital. Shenzhen city has established a preexisting Infectious Disease Epidemic Plan IDEP , which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital.", "Shenzhen city has established a preexisting Infectious Disease Epidemic Plan IDEP , which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital. Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds. 2019-nCoV patients should be admitted to singlebedded, negative pressure rooms in isolated units with intensive care and monitoring . Clinical engineering should have plans to reconstruct standard rooms . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution.", "Clinical engineering should have plans to reconstruct standard rooms . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution. Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors . More importantly, because the hospitals' ability to respond to the outbreak largely depends on their available ICU beds, the plan to increase ICU bed capacity needs to be determined. Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 95% CI, 1.4 to 3.9 , or as high as between 3.6 and 4.0 .", "Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 95% CI, 1.4 to 3.9 , or as high as between 3.6 and 4.0 . The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible . The higher viral load and aerosol-generating procedures, such as noninvasive ventilation, magnify the exposure and transmission risk . Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator.", "Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator. Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee CNHC . In addition, wellequipped fever clinic as triage station with trained staff knowing 2019-nCoV case definitions is established. For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens , and implementing droplet isolation to break the transmission chain in the healthcare setting . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers .", "For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens , and implementing droplet isolation to break the transmission chain in the healthcare setting . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers . The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020. Psychotherapists have also been invited to join medical teams to evaluate and deal with potential stress and depression for the safety of the healthcare workers. Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation ECMO according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged.", "Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation ECMO according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged. These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality. On the contrary, the previously reported mortality of 2019-nCoV patients in Wuhan ranged from 11 to 15% ." ]

[ "nan Text: The main challenge may include . early identification of outbreak, . rapid expansion of patients, . high risk of nosocomial transmission, . unpredictability of size impacted, and . lack of backup resource. These challenges have caused severe shortage of healthcare workers, medical materials, and beds with isolation. The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage.", "The Spring Festival holiday has greatly aggravated the shortage of human resources and heavy traffic flow due to the vacation of healthy workers and factory workers, which further magnified the risk of transmission. The key point is to discriminate the infectious disease outbreak from regular clustering cases of flu-like diseases at early stage. There is a trade-off between false alarm causing population panic and delayed identification leading to social crisis. Early identification of 2019-nCoV infection presents a major challenge for the frontline clinicians. Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever 98% , cough 76% , and diarrhea 3% , often more severe in older adults with pre-existing chronic comorbidities . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia .", "Its clinical symptoms largely overlap with those of common acute respiratory illnesses, including fever 98% , cough 76% , and diarrhea 3% , often more severe in older adults with pre-existing chronic comorbidities . Usually, the laboratory abnormalities include lymphocytopenia and hypoxemia . The initial chest radiographs may vary from minimal abnormality to bilateral ground-glass opacity or subsegmental areas of consolidation . In addition, asymptomatic cases and lack of diagnosis kits result in delayed or even missed diagnosis inevitable and makes many other patients, visitors, and healthcare workers exposed to the 2019-nCoV infection. Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital IDH .", "Critical care response to the outbreak of coronavirus should happen not only at the level of hospital, but also at the level of the city which is dominated by the government. At the early stage, the size of the patients' population is not beyond the capability of local infectious diseases hospital IDH . The general hospital is responsible for fever triage, identifying suspected cases, and transferring to the local IDH. Such a plan is mandatory for every hospital. Shenzhen city has established a preexisting Infectious Disease Epidemic Plan IDEP , which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital.", "Shenzhen city has established a preexisting Infectious Disease Epidemic Plan IDEP , which has facilitated managing and containing local outbreak of the 2019-nCoV. In case the patient load exceeds the hospital capability of the IDH, new IDHs should be considered either by building a temporary new IDH or reconstructing an existing hospital. Wuhan, the epicenter of the outbreak, is racing against time to build two specialized hospitals for nCoV patients, namely Huoshenshan and Leishenshan hospital, whereas a different strategy has been undertaken in Shenzhen city by reconstructing an existing hospital to become an IDH with capability of 800 beds. 2019-nCoV patients should be admitted to singlebedded, negative pressure rooms in isolated units with intensive care and monitoring . Clinical engineering should have plans to reconstruct standard rooms . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution.", "Clinical engineering should have plans to reconstruct standard rooms . Retrofitting the rooms with externally exhausted HEPA filters may be an expedient solution. Also, the general hospital may consider procedures such as suspending elective surgeries, canceling ambulatory clinics and outpatient diagnostic procedures, transferring patients to other institutions, and restricting hospital visitors . More importantly, because the hospitals' ability to respond to the outbreak largely depends on their available ICU beds, the plan to increase ICU bed capacity needs to be determined. Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 95% CI, 1.4 to 3.9 , or as high as between 3.6 and 4.0 .", "Caring for 2019-nCoV patients represents a substantial exposure risk for ICU staff because of the following reasons: highly contagious with multiple transmission route, high exposure dose, long daily contact hours, and ICU stay. The basic reproductive number was estimated to be 2.2 95% CI, 1.4 to 3.9 , or as high as between 3.6 and 4.0 . The 2019-nCoV is proved to be transmitted by respiratory droplets, contact, and fecal-oral, even transmission through the eye is possible . The higher viral load and aerosol-generating procedures, such as noninvasive ventilation, magnify the exposure and transmission risk . Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator.", "Moreover, virus shedding can be prolonged and last for > 3 weeks according to some literature and our unpublished data . Healthcare providers and those in contact with infected patients should utilize contact, droplet, and airborne precautions with N95 respirator. Strict infection prevention and control practices have been implemented and audited in our units following the infection prevention and control plan published by China's National Health Committee CNHC . In addition, wellequipped fever clinic as triage station with trained staff knowing 2019-nCoV case definitions is established. For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens , and implementing droplet isolation to break the transmission chain in the healthcare setting . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers .", "For suspected 2019-nCoV infection, several key points are crucial procedures: recording a detailed history, standardizing pneumonia workup, obtaining lower respiratory tract specimens , and implementing droplet isolation to break the transmission chain in the healthcare setting . The risk of 2019-nCoV exposure may cause significant psychosocial stress on healthcare workers . The death of a retired ENT physician from a 2019-nCoV infection has added to fears in January 2020. Psychotherapists have also been invited to join medical teams to evaluate and deal with potential stress and depression for the safety of the healthcare workers. Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation ECMO according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged.", "Critical management 2019-nCoV management was largely supportive, including intubation, early prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation ECMO according to the recommendations updated by CNHC. Low-dose systematic corticosteroids, lopinavir/ritonavir, and atomization inhalation of interferon were encouraged. These critical managements have worked well so far, as our 2019-nCoV patients had zero mortality. On the contrary, the previously reported mortality of 2019-nCoV patients in Wuhan ranged from 11 to 15% ." ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "nan Text: The emergence and outbreak of a newly discovered acute respiratory disease in Wuhan, China, has affected greater than 40,000 people, and killed more than 1,000 as of Feb. 10, 2020. A new human coronavirus, SARS-CoV-2, was quickly identified, and the associated disease is now referred to as coronavirus disease discovered in 2019 COVID-19 com/novel-coronavirus-covid-19-portal/ . According to what has been reported , COVID-2019 seems to have similar clinical manifestations to that of the severe acute respiratory syndrome SARS caused by SARS-CoV. The SARS-CoV-2 genome sequence also has ∼80% identity with SARS-CoV, but it is most similar to some bat beta-coronaviruses, with the highest being >96% identity . Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 .", "Currently, there are speculations, rumours and conspiracy theories that SARS-CoV-2 is of laboratory origin. Some people have alleged that the human SARS-CoV-2 was leaked directly from a laboratory in Wuhan where a bat CoV RaTG13 was recently reported, which shared ∼96% homology with the SARS-CoV-2 . However, as we know, the human SARS-CoV and intermediate host palm civet SARSlike CoV shared 99.8% homology, with a total of 202 single-nucleotide nt variations SNVs identified across the genome . Given that there are greater than 1,100 nt differences between the human SARS-CoV-2 and the bat RaTG13-CoV , which are distributed throughout the genome in a naturally occurring pattern following the evolutionary characteristics typical of CoVs, it is highly unlikely that RaTG13 CoV is the immediate source of SARS-CoV-2. The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2.", "The absence of a logical targeted pattern in the new viral sequences and a close relative in a wildlife species bats are the most revealing signs that SARS-CoV-2 evolved by natural evolution. A search for an intermediate animal host between bats and humans is needed to identify animal CoVs more closely related to human SARS-CoV-2. There is speculation that pangolins might carry CoVs closely related to SARS-CoV-2, but the data to substantiate this is not yet published Another claim in Chinese social media points to a Nature Medicine paper published in 2015 , which reports the construction of a chimeric CoV with a bat CoV S gene SHC014 in the backbone of a SARS CoV that has adapted to infect mice MA15 and is capable of infecting human cells . However, this claim lacks any scientific basis and must be discounted because of significant divergence in the genetic sequence of this construct with the new SARS-CoV-2 >5,000 nucleotides . The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation.", "The mouse-adapted SARS virus MA15 was generated by serial passage of an infectious wildtype SARS CoV clone in the respiratory tract of BALB/c mice. After 15 passages in mice, the SARS-CoV gained elevated replication and lung pathogenesis in aged mice hence M15 , due to six coding genetic mutations associated with mouse adaptation. It is likely that MA15 is highly attenuated to replicate in human cells or patients due to the mouse adaptation. It was proposed that the S gene from bat-derived CoV, unlike that from human patients-or civetsderived viruses, was unable to use human ACE2 as a receptor for entry into human cells . Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry .", "Civets were proposed to be an intermediate host of the bat-CoVs, capable of spreading SARS CoV to humans . However, in 2013 several novel bat coronaviruses were isolated from Chinese horseshoe bats and the bat SARS-like or SL-CoV-WIV1 was able to use ACE2 from humans, civets and Chinese horseshoe bats for entry . Combined with evolutionary evidence that the bat ACE2 gene has been positively selected at the same contact sites as the human ACE2 gene for interacting with SARS CoV , it was proposed that an intermediate host may not be necessary and that some bat SL-CoVs may be able to directly infect human hosts. To directly address this possibility, the exact S gene from bat coronavirus SL-SHC014 was synthesized and used to generate a chimeric virus in the mouse adapted MA15 SARS-CoV backbone. The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice .", "The resultant SL-SHC014-MA15 virus could indeed efficiently use human ACE2 and replicate in primary human airway cells to similar titres as epidemic strains of SARS-CoV. While SL-SHC014-MA15 can replicate efficiently in young and aged mouse lungs, infection was attenuated, and less virus antigen was present in the airway epithelium as compared to SARS MA15, which causes lethal outcomes in aged mice . Due to the elevated pathogenic activity of the SHC014-MA15 chimeric virus relative to MA15 chimeric virus with the original human SARS S gene in mice, such experiments with SL-SHC014-MA15 chimeric virus were later restricted as gain of function GOF studies under the US government-mandated pause policy The current COVID-2019 epidemic has restarted the debate over the risks of constructing such viruses that could have pandemic potential, irrespective of the finding that these bat CoVs already exist in nature. Regardless, upon careful phylogenetic analyses by multiple international groups , the SARS-CoV-2 is undoubtedly distinct from SL-SHC014-MA15, with >6,000 nucleotide differences across the whole genome. Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory.", "Therefore, once again there is no credible evidence to support the claim that the SARS-CoV-2 is derived from the chimeric SL-SHC014-MA15 virus. There are also rumours that the SARS-CoV-2 was artificially, or intentionally, made by humans in the lab, and this is highlighted in one manuscript submitted to BioRxiv a manuscript sharing site prior to any peer review , claiming that SARS-CoV-2 has HIV sequence in it and was thus likely generated in the laboratory. In a rebuttal paper led by an HIV-1 virologist Dr. Feng Gao, they used careful bioinformatics analyses to demonstrate that the original claim of multiple HIV insertions into the SARS-CoV-2 is not HIV-1 specific but random . Because of the many concerns raised by the international community, the authors who made the initial claim have already withdrawn this report. Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV.", "Evolution is stepwise and accrues mutations gradually over time, whereas synthetic constructs would typically use a known backbone and introduce logical or targeted changes instead of the randomly occurring mutations that are present in naturally isolated viruses such as bat CoV RaTG13. In our view, there is currently no credible evidence to support the claim that SARS-CoV-2 originated from a laboratory-engineered CoV. It is more likely that SARS-CoV-2 is a recombinant CoV generated in nature between a bat CoV and another coronavirus in an intermediate animal host. More studies are needed to explore this possibility and resolve the natural origin of SARS-CoV-2. We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s .", "We should emphasize that, although SARS-CoV-2 shows no evidence of laboratory origin, viruses with such great public health threats must be handled properly in the laboratory and also properly regulated by the scientific community and governments. No potential conflict of interest was reported by the author s . Susan R. Weiss" ]

[ "The objective of this clinical trial was to evaluate the effectiveness of zinc supplementation on diarrhea and average daily weight gain ADG in pre-weaned dairy calves. A total of 1,482 healthy Holstein heifer and bull calves from a large California dairy were enrolled at 24 to 48 hours of age until hutch exit at approximately 90 days of age. Calves were block-randomized by time to one of three treatments: 1 placebo, 2 zinc methionine ZM , or 3 zinc sulfate ZS administered in milk once daily for 14 days. Serum total protein at enrollment and body weight at birth, treatment end, and hutch exit were measured. Fecal consistency was assessed daily for 28 days post-enrollment. For a random sample of 127 calves, serum zinc concentrations before and after treatment and a fecal antigen ELISA at diarrhea start and resolution for Escherichia coli K99, rotavirus, coronavirus, and Cryptosporidium parvum were performed.", "Fecal consistency was assessed daily for 28 days post-enrollment. For a random sample of 127 calves, serum zinc concentrations before and after treatment and a fecal antigen ELISA at diarrhea start and resolution for Escherichia coli K99, rotavirus, coronavirus, and Cryptosporidium parvum were performed. Linear regression showed that ZM-treated bull calves had 22 g increased ADG compared to placebo-treated bulls P = 0.042 . ZM-treated heifers had 9 g decreased ADG compared to placebo-treated heifers P = 0.037 , after adjusting for average birth weight. Sex-stratified models showed that high birth weight heifers treated with ZM gained more than placebo-treated heifers of the same birth weight, which suggests a dose-response effect rather than a true sex-specific effect of ZM on ADG. Cox regression showed that ZM and ZS-treated calves had a 14.7% P = 0.015 and 13.9% P = 0.022 reduced hazard of diarrhea, respectively, compared to placebo-treated calves.", "Sex-stratified models showed that high birth weight heifers treated with ZM gained more than placebo-treated heifers of the same birth weight, which suggests a dose-response effect rather than a true sex-specific effect of ZM on ADG. Cox regression showed that ZM and ZS-treated calves had a 14.7% P = 0.015 and 13.9% P = 0.022 reduced hazard of diarrhea, respectively, compared to placebo-treated calves. Calves supplemented for at least the first five days of diarrhea with ZM and ZS had a 21.4% P = 0.027 and 13.0% P = 0.040 increased hazard of cure from diarrhea, respectively, compared to placebo-treated calves. Logistic regression showed that the odds of microbiological cure at diarrhea resolution for rotavirus, C. parvum, or any single fecal pathogen was not different between treatment groups. Zinc supplementation delayed diarrhea and expedited diarrhea recovery in pre-weaned calves. Additionally, zinc improved weight gain differentially in bulls compared to heifers, indicating a research need for sex-specific dosing.", "Zinc supplementation delayed diarrhea and expedited diarrhea recovery in pre-weaned calves. Additionally, zinc improved weight gain differentially in bulls compared to heifers, indicating a research need for sex-specific dosing. Text: Introduction Diarrhea is the leading cause of morbidity and mortality and the most common reason for antimicrobial drug treatments in pre-weaned dairy heifers . A USDA survey of preweaned dairy heifers reported that 24% experienced diarrhea and 18% received antimicrobial treatment for it . Diarrhea is also a leading cause of morbidity and the second foremost cause of mortality in children with over 1 billion cases and a half a million deaths annually . Zinc supplementation in children decreases the incidence, duration, and severity of diarrhea, increases recovery rates, decreases the use of antibiotics and antidiarrheal medications, and reduces mortality .", "Diarrhea is also a leading cause of morbidity and the second foremost cause of mortality in children with over 1 billion cases and a half a million deaths annually . Zinc supplementation in children decreases the incidence, duration, and severity of diarrhea, increases recovery rates, decreases the use of antibiotics and antidiarrheal medications, and reduces mortality . In a clinical trial that established a non-toxic zinc dose and investigated its therapeutic use for diarrhea in neonatal dairy calves, zinc-treated calves had numerically quicker clinical recovery, increased weight gain, and higher odds of fecal clearance of Cryptosporidium parvum between diarrhea onset and recovery compared with placebotreated calves . As a result, zinc supplementation may be beneficial for prevention of diarrhea in dairy calves and, thus, minimize antimicrobial use. However, studies investigating zinc's potential effectiveness are lacking. In children, both organic zinc acetate, zinc gluconate, and zinc methionine and inorganic zinc sulfate and zinc oxide zinc formulations are beneficial in the prevention and treatment of childhood diarrhea .", "However, studies investigating zinc's potential effectiveness are lacking. In children, both organic zinc acetate, zinc gluconate, and zinc methionine and inorganic zinc sulfate and zinc oxide zinc formulations are beneficial in the prevention and treatment of childhood diarrhea . However, differing bioavailability was observed in several animal studies . In addition, the underlying mechanism of action of oral zinc is unknown . Hence, contrasting the effect, if any, of organic compared to inorganic zinc formulations in pre-weaned calves may help identify differences in mode of action. The objective of this clinical trial was to compare average daily weight gain ADG and the incidence and duration of diarrhea in pre-weaned dairy calves randomly assigned to receive either organic zinc methionine ZM , inorganic zinc sulfate ZS , or a placebo in milk once daily for 14 days.", "Hence, contrasting the effect, if any, of organic compared to inorganic zinc formulations in pre-weaned calves may help identify differences in mode of action. The objective of this clinical trial was to compare average daily weight gain ADG and the incidence and duration of diarrhea in pre-weaned dairy calves randomly assigned to receive either organic zinc methionine ZM , inorganic zinc sulfate ZS , or a placebo in milk once daily for 14 days. By elucidating the potential role of zinc supplementation in prevention of diarrhea in preweaned dairy calves, calf morbidity, mortality, and antimicrobial usage may be mitigated. A double-blind, block randomized, placebo-controlled clinical trial was conducted between December 14, 2015 and June 15, 2016 on a large dairy in California's San Joaquin Valley. The dairy was selected based on the owner and calf manager's willingness to participate in the study. The dairy herd was composed of 5,500 lactating cows, predominantly Holsteins, and housed approximately 1,600 pre-weaned calves.", "The dairy was selected based on the owner and calf manager's willingness to participate in the study. The dairy herd was composed of 5,500 lactating cows, predominantly Holsteins, and housed approximately 1,600 pre-weaned calves. Approximately 75% of the calves were born on the participating dairy and 25% were born on an affiliated dairy located approximately 10 miles away. Calves enrolled in the trial included healthy Holstein heifer or bull calves 24 to 48 hours of age. Calves were determined to be healthy via visual examination by a veterinarian HF or a trained researcher. Calves were excluded if they had obvious morbidities or congenital defects, were non-Holstein, born on the affiliated dairy, younger than 24 hours of age or older than 48 hours of age at the time of enrollment.", "Calves were determined to be healthy via visual examination by a veterinarian HF or a trained researcher. Calves were excluded if they had obvious morbidities or congenital defects, were non-Holstein, born on the affiliated dairy, younger than 24 hours of age or older than 48 hours of age at the time of enrollment. Calves from the affiliated dairy were excluded due to differences in physical location and management practices of pre-partum cows. All procedures were approved by the University of California Davis Institutional Animal Care and Use Committee protocol number 18067 Approved: March 6, 2014 . 107 g between treatment groups Stata, College Station, TX . After allowing for 15% attrition and assuming 50% incidence of diarrhea based on study authors expert opinion, and a difference in ADG of 107g , a sample size of 500 calves per treatment group n = 1500 total was deemed required.", "107 g between treatment groups Stata, College Station, TX . After allowing for 15% attrition and assuming 50% incidence of diarrhea based on study authors expert opinion, and a difference in ADG of 107g , a sample size of 500 calves per treatment group n = 1500 total was deemed required. Newborn calves were removed from the dam within an hour of birth and placed in a strawbedded, group calf pen where their navels were dipped in an iodine-based solution. Each calf received 4 liters of colostrum within 1 hour of birth and a second colostrum feeding 2 liters 6-10 hours after birth. Colostrum was refrigerated for < 48 hours and heated in a hot water bath prior to feeding using an esophageal tube feeder. Within 18 hours of birth, pre-weaned calves were transported to individual metal hutches initially bedded with almond shells.", "Colostrum was refrigerated for < 48 hours and heated in a hot water bath prior to feeding using an esophageal tube feeder. Within 18 hours of birth, pre-weaned calves were transported to individual metal hutches initially bedded with almond shells. Straw hay was later added to wet and muddy hutches throughout the pre-weaning period. For the first 14 days of life, pre-weaned calves were bottle-fed 1.9 liters of milk twice daily and 1.9 liters of a commercial oral electrolyte solution Calva Lyte; Calva Products, Inc., Acampo, CA once daily between milk feedings. Milk consisted of a combination of pasteurized waste milk, rehydrated commercial milk replacer powder Strauss Feeds LLC, Watertown, WI , tetracycline and neomycin powder, and additional supplements S1 Table . The proportion of pasteurized waste milk to milk replacer varied with each feeding, as the volume of waste milk varied with changes in the number and production of cows contributing to the waste milk tank.", "Milk consisted of a combination of pasteurized waste milk, rehydrated commercial milk replacer powder Strauss Feeds LLC, Watertown, WI , tetracycline and neomycin powder, and additional supplements S1 Table . The proportion of pasteurized waste milk to milk replacer varied with each feeding, as the volume of waste milk varied with changes in the number and production of cows contributing to the waste milk tank. After 14 days of age, calves were bottle-fed 2.8 liters of milk twice daily. Calves with clinical diarrhea received 1.9 liters of a commercial oral electrolyte solution NuLife; Genex Cooperative, Inc., Shawano, WI once daily between milk feedings. A list of ingredients that make up the two oral electrolyte solutions can be found in S2 Table. All pre-weaned calves had free choice access to water and a calf starter grain mix.", "A list of ingredients that make up the two oral electrolyte solutions can be found in S2 Table. All pre-weaned calves had free choice access to water and a calf starter grain mix. Calves were gradually weaned over a 10-day period, starting at approximately 60 days of age after which calves received a grower grain mix until 90 days of age when they were moved to group pens. Each calf received 1 mL of a selenium supplement MU-SE; Merck Animal Health, Boxmeer, Netherlands intramuscularly within 24 hours of age and an intranasal vaccine Inforce 3, Zoetis, Inc., Florham Park, NJ within 48 hours of age and again near the time of weaning. Approximately 8.3% n = 126 of all enrolled calves were also vaccinated using an autogenous Moraxella bovis/bovoculi bacterin vaccine Newport Laboratories, Inc., Worthington, MN for the prevention of pinkeye at 5 and 7 weeks of age. All pre-weaned calves were evaluated daily by dairy personnel for calfhood diseases and treated according to standard on-farm treatment protocols.", "Approximately 8.3% n = 126 of all enrolled calves were also vaccinated using an autogenous Moraxella bovis/bovoculi bacterin vaccine Newport Laboratories, Inc., Worthington, MN for the prevention of pinkeye at 5 and 7 weeks of age. All pre-weaned calves were evaluated daily by dairy personnel for calfhood diseases and treated according to standard on-farm treatment protocols. With regard to diarrhea therapy, calves less than two weeks of age with clinical diarrhea received an oral mixture of 118.5 mL 2.08 g bismuth subsalicylate Bismusal Suspension, Durvet, Inc., Blue Springs, MO and 31.5 mL 1575 mg spectinomycin SpectoGard, Bimeda, Inc., Le Sueur, MN once daily for two days. Calves older than two weeks of age with clinical diarrhea received oral sulfamethoxazole 1600 mg /trimethoprim 320 mg Amneal Pharmaceuticls of NY, Hauppauge, NY once daily for 2 to 3 days. Repeated treatment was at the discretion of the calf manager. inductively coupled plasma mass spectrometry .", "Repeated treatment was at the discretion of the calf manager. inductively coupled plasma mass spectrometry . Financial limitations restricted the ability to test more than two samples of each dietary component. Due to variation of zinc content in duplicate samples, the maximum concentration was used to estimate the daily zinc intake during the first 14 days of life S3 Table . In blocks of 36, enrolled calves were randomized using a random number generator Microsoft Corporation, Redmond, WA to one of three treatment groups: 1 placebo, 2 ZM, or 3 ZS to be administered in the morning milk feeding once daily for 14 days, starting on the day after enrollment. During the 14-day zinc treatment period, study calves that did not drink the entire milk bottle were tube-fed the remaining milk by trained technicians using an esophageal feeder disinfected between uses.", "In blocks of 36, enrolled calves were randomized using a random number generator Microsoft Corporation, Redmond, WA to one of three treatment groups: 1 placebo, 2 ZM, or 3 ZS to be administered in the morning milk feeding once daily for 14 days, starting on the day after enrollment. During the 14-day zinc treatment period, study calves that did not drink the entire milk bottle were tube-fed the remaining milk by trained technicians using an esophageal feeder disinfected between uses. The ZM treatment group received 0.45 g zinc methionine complex equivalent to 80 mg of elemental zinc as the product Zinpro180 Zinpro Corporation, Eden Prairie, MN combined with 0.44 g milk replacer powder. The ZS treatment group received 0.22 g zinc sulfate monohydrate equivalent to 80 mg of elemental zinc Sigma-Aldrich Company, St. Louis, MO combined with 0.44 g milk replacer powder. The placebo treatment group received approximately 0.44 g fresh milk replacer powder. Zinc supplementation was based on a previously published clinical trial, toxicological studies, and nutritional guidelines 11, .", "The placebo treatment group received approximately 0.44 g fresh milk replacer powder. Zinc supplementation was based on a previously published clinical trial, toxicological studies, and nutritional guidelines 11, . The milk replacer powder used in treatment preparation was the same product used in the pre-weaned calf milk ration. Treatments were weighed GX-2000 precision scale; A&D Co Ltd., San Jose, CA at the Dairy Epidemiology Laboratory at the University of California, Davis Veterinary Medicine Teaching and Research Center VMTRC; Aly Lab in Tulare, CA and placed in 2.0 mL microcentrifuge tubes with polypropylene snap caps Fisher Scientific, Pittsburgh, PA . Prior to study commencement, a color was randomly and permanently assigned to each treatment group, after which treatment tubes, calf milk bottles and calf hutches were marked with either pink, orange, or yellow ink. The study investigators and technicians responsible for treatment preparation, allocation, administration and data collection were blinded to the color assignment until completion of the trial.", "Prior to study commencement, a color was randomly and permanently assigned to each treatment group, after which treatment tubes, calf milk bottles and calf hutches were marked with either pink, orange, or yellow ink. The study investigators and technicians responsible for treatment preparation, allocation, administration and data collection were blinded to the color assignment until completion of the trial. For each calf, the study period started at enrollment 24 to 48 hours of age and ended when the calf exited the hutch approximately 90 days of age and from here onwards will be referred to as the \"pre-weaning period.\" Calf enrollment and study procedures were performed daily at the time of morning feeding. At enrollment, calf characteristics, including sex, birth date, time of first colostrum feeding, and treatment color were recorded. Attitude and feces were assessed daily until 28 days post-enrollment using previously published methods by two study investigators, a veterinarian and a trained researcher.", "At enrollment, calf characteristics, including sex, birth date, time of first colostrum feeding, and treatment color were recorded. Attitude and feces were assessed daily until 28 days post-enrollment using previously published methods by two study investigators, a veterinarian and a trained researcher. Attitude scoring was based on a threepoint scale. A calf with an attitude score of 1 was bright, alert, and readily stood with stimulation; a calf with a score of 2 was quiet, alert, and stood only with moderate stimulation; a calf with a score of 3 exhibited a dull mentation and remained recumbent in response to stimulation. Fecal scoring was performed only on fresh feces and was also based on a three-point scale, as 1 solid , 2 semi-formed/loose , or 3 watery . If no fresh feces were observed in the hutch, \"none seen\" NS was recorded.", "Fecal scoring was performed only on fresh feces and was also based on a three-point scale, as 1 solid , 2 semi-formed/loose , or 3 watery . If no fresh feces were observed in the hutch, \"none seen\" NS was recorded. Body weight was measured using a digital scale at birth, end of treatment, and hutch exit by farm employees with the exception of end of treatment weights, which were recorded by study investigators. Treatments for farm-diagnosed illnesses were performed and recorded on hutch cards by the calf manager. Study investigators regularly recorded this information from cards in addition to extracting treatment event reports from DairyComp 305 Valley Agricultural Software, Tulare, CA . Though daily diagnosis and treatment of study calves was performed and recorded by the calf manager, a veterinarian was responsible for examining and determining whether study calves met specific criteria for euthanasia.", "Study investigators regularly recorded this information from cards in addition to extracting treatment event reports from DairyComp 305 Valley Agricultural Software, Tulare, CA . Though daily diagnosis and treatment of study calves was performed and recorded by the calf manager, a veterinarian was responsible for examining and determining whether study calves met specific criteria for euthanasia. A calf was euthanized if morbidity was severe enough to significantly depress appetite, hydration status, attitude, mentation, and/or ambulatory capability and the calf showed limited to no immediate response to therapy or supportive care. Study calves were euthanized by the calf manager using an on-farm captive bolt protocol established by the herd veterinarian within 3 hours of the decision to euthanize. Enrolled calves that died prior to hutch exit were necropsied within 24 hours of death by a veterinarian. All calves were monitored throughout the study period for evidence of zinc toxicity.", "Enrolled calves that died prior to hutch exit were necropsied within 24 hours of death by a veterinarian. All calves were monitored throughout the study period for evidence of zinc toxicity. At the end of the study period, calves were cared for at the dairy in accordance with standard commercial operations. Using the same random number generator, a random sample of 127 calves was selected for additional biologic sampling. Approximately 8 to 10% of the study population was selected due to the financial constraints of additional laboratory testing. Serum zinc concentration at baseline and in response to treatment were evaluated for the three treatment groups.", "Approximately 8 to 10% of the study population was selected due to the financial constraints of additional laboratory testing. Serum zinc concentration at baseline and in response to treatment were evaluated for the three treatment groups. Feces collected on the first day of diarrhea and at diarrhea resolution were evaluated for four fecal pathogens Escherichia. coli K99, bovine rotavirus and coronavirus, and Cryptosporidium parvum oocysts . Serum total protein. At enrollment, blood from each calf was collected from the jugular vein using a 20 gauge 1-inch multi-sample needle Exelint International Co., Redondo Beach, CA and placed into a 10 mL red top serum tube BD Vacutainer, Franklin Lakes, NJ for determination of total protein.", "Serum total protein. At enrollment, blood from each calf was collected from the jugular vein using a 20 gauge 1-inch multi-sample needle Exelint International Co., Redondo Beach, CA and placed into a 10 mL red top serum tube BD Vacutainer, Franklin Lakes, NJ for determination of total protein. Samples remained at room temperature for up to 12 hours until clotting and were then centrifuged International Equipment Company, CRU-5000, Needham Heights, MA for 15 minutes. Total protein g/dL was measured by a single investigator HF on decanted serum using a handheld refractometer Sper Scientific, Model 300005, Scottsdale, AZ . Serum zinc. For the 127 randomly-sampled calves, additional blood was collected at enrollment and on the last day of treatment, as described above, and placed into 6.0 mL trace element tubes BD Vacutainer, Franklin Lakes, NJ .", "Serum zinc. For the 127 randomly-sampled calves, additional blood was collected at enrollment and on the last day of treatment, as described above, and placed into 6.0 mL trace element tubes BD Vacutainer, Franklin Lakes, NJ . Serum was extracted, as described above, and placed in a 2.0 mL microcentrifuge tube with a polypropylene snap cap Fisher Scientific, Pittsburgh, PA and stored at −20˚C until analysis. Using the same random number generator, 36 of the 127 sampled calves were randomly selected for analysis due to limited financial resources. The pre-and post-zinc supplementation serum samples from each calf n = 72 were analyzed for zinc concentration ppm by ICP-OES inductively coupled plasma-optical emission spectroscopy at the CAHFS Laboratory. Quality control samples, including method blanks, laboratory control spikes, and reference Sigma serum, were run with each set of study samples.", "The pre-and post-zinc supplementation serum samples from each calf n = 72 were analyzed for zinc concentration ppm by ICP-OES inductively coupled plasma-optical emission spectroscopy at the CAHFS Laboratory. Quality control samples, including method blanks, laboratory control spikes, and reference Sigma serum, were run with each set of study samples. Fecal analysis. For 127 randomly-sampled calves at the first diarrhea episode, fecal samples were collected at two time points, the first day of diarrhea fecal score > 1 and the day diarrhea resolved second day of fecal score = 1 . Using new gloves and sterile lubricant, fresh feces was collected by digital rectal stimulation into 20 mL polypropylene twist-top jars The Cary Company, Addison, IL and stored at -20˚C until analysis. Fecal samples were tested at the Dairy Epidemiology Laboratory, VMTRC by a veterinarian for E. coli K99, bovine rotavirus and coronavirus, and C. parvum oocysts using a commercial kit Pathasure Enteritis 4; Biovet, Quebec, Canada that is highly specific > 90% and sensitive E. coli K99, 93%; rotavirus, 100%; coronavirus, 77% .", "Using new gloves and sterile lubricant, fresh feces was collected by digital rectal stimulation into 20 mL polypropylene twist-top jars The Cary Company, Addison, IL and stored at -20˚C until analysis. Fecal samples were tested at the Dairy Epidemiology Laboratory, VMTRC by a veterinarian for E. coli K99, bovine rotavirus and coronavirus, and C. parvum oocysts using a commercial kit Pathasure Enteritis 4; Biovet, Quebec, Canada that is highly specific > 90% and sensitive E. coli K99, 93%; rotavirus, 100%; coronavirus, 77% . For calves with a first-day diarrhea sample on or before 7 days of age, both fecal samples were tested for all four pathogens. For calves with a first-day diarrhea sample after 7 days of age, both fecal samples were tested for three pathogens C. parvum, bovine rotavirus and coronavirus . Samples from calves older than 7 days of age were not tested for E. coli K99 based on calves' susceptibility . Testing was performed according to kit manufacturer guidelines, and a low-temperature incubator Fisher Scientific, Model 146, Pittsburgh, PA was used during incubation periods.", "Samples from calves older than 7 days of age were not tested for E. coli K99 based on calves' susceptibility . Testing was performed according to kit manufacturer guidelines, and a low-temperature incubator Fisher Scientific, Model 146, Pittsburgh, PA was used during incubation periods. Test results were recorded as positive or negative using control wells for color comparison. If the color change was darker than the negative control, the sample was considered positive. Milk zinc. For each of the 107 study days December 15, 2015 to March 31, 2016 of zinc supplementation, approximately 1.5 mL of treated milk from two bottles of each treatment group were randomly collected into 2.0 mL microcentrifuge tubes with polypropylene snap caps Fisher Scientific, Pittsburgh, PA , and stored at −20˚C until analysis.", "Milk zinc. For each of the 107 study days December 15, 2015 to March 31, 2016 of zinc supplementation, approximately 1.5 mL of treated milk from two bottles of each treatment group were randomly collected into 2.0 mL microcentrifuge tubes with polypropylene snap caps Fisher Scientific, Pittsburgh, PA , and stored at −20˚C until analysis. At the time of analysis, milk samples were thawed at 4˚C, vortexed, pooled by week and treatment group, and analyzed for zinc concentration ppm by ICP-MS inductively coupled plasma mass spectrometry at the CAHFS Laboratory. Quality control samples, including method blanks, laboratory control spikes, National Institute of Standards and Technology NIST reference materials NIST 1640 , and a spiked milk sample, were run with each set of study samples. Data analysis was performed using R Statistic Software version 3.3.1 and Stata IC 14.2 College Station, TX . Statistical differences were determined at the 5% level of significance using per protocol analysis.", "Data analysis was performed using R Statistic Software version 3.3.1 and Stata IC 14.2 College Station, TX . Statistical differences were determined at the 5% level of significance using per protocol analysis. An ANOVA was used to compare calves in each treatment group at enrollment with respect to birth weight kg , serum total protein g/dL , attitude score, and fecal score. Oral zinc dose at the start and end of treatment was calculated as the zinc supplementation dose 80 mg divided by calf body weight kg at birth and on the last day of treatment, respectively. An ANOVA was used to compare oral zinc dose mg/kg at treatment start and end as well as mean body weight kg at birth, end of treatment, and hutch exit between treatment groups and between bulls and heifers. A Chi-Square test of Independence was used to compare the proportions of calves by sex as well as mortality between treatment groups.", "An ANOVA was used to compare oral zinc dose mg/kg at treatment start and end as well as mean body weight kg at birth, end of treatment, and hutch exit between treatment groups and between bulls and heifers. A Chi-Square test of Independence was used to compare the proportions of calves by sex as well as mortality between treatment groups. For all analyses, Tukey's Honest Significant Difference method was used to generate pairwise comparisons to further characterize significant differences identified by ANOVA. Residual diagnostics, including Residuals vs. Fitted, Scale-Location, Normal Q-Q, and Cook's distances plots, were used to validate all ANOVA model assumptions. The non-parametric Kruskal-Wallis Rank Sum test was used when assumptions were violated. For the randomly-sampled calves, Fisher exact tests were used to compare fecal pathogen prevalence on the first day of diarrhea and at diarrhea resolution between treatment groups.", "The non-parametric Kruskal-Wallis Rank Sum test was used when assumptions were violated. For the randomly-sampled calves, Fisher exact tests were used to compare fecal pathogen prevalence on the first day of diarrhea and at diarrhea resolution between treatment groups. Pairwise comparisons with Bonferroni adjustment were used to identify specific differences in pathogen prevalence. An ANOVA was used to compare serum zinc concentration before and after treatment between treatment groups and between bulls and heifers. A Kruskal-Wallis Rank Sum test was used to compare rank sums of zinc concentrations in pooled milk samples from different treatment groups. Post-hoc Nemenyi-tests for pairwise multiple comparisons of ranked data were used to identify specific differences in zinc concentrations between groups.", "A Kruskal-Wallis Rank Sum test was used to compare rank sums of zinc concentrations in pooled milk samples from different treatment groups. Post-hoc Nemenyi-tests for pairwise multiple comparisons of ranked data were used to identify specific differences in zinc concentrations between groups. For all regression models in this study, univariate regression was first used to evaluate associations between individual predictor variables and outcomes. All variables with statistical and/or biological significance were initially included in multivariate regression models. The final models were built using a manual backwards elimination procedure, with a significance level of P > 0.05 as the removal criterion. Confounding was assessed using the method of change of estimates, where a 10% or greater change in the estimate of the treatment group regression coefficient between the models with and without the confounder variable was used as evidence of confounding .", "The final models were built using a manual backwards elimination procedure, with a significance level of P > 0.05 as the removal criterion. Confounding was assessed using the method of change of estimates, where a 10% or greater change in the estimate of the treatment group regression coefficient between the models with and without the confounder variable was used as evidence of confounding . Variables identified as confounders were included in the final model. All possible interactions between treatment group and predictor variables were explored and retained in the final model if statistically significant. Microbiological cure. For the randomly-sampled calves, logistic regression was used to evaluate associations between microbiological cure and treatment group.", "Microbiological cure. For the randomly-sampled calves, logistic regression was used to evaluate associations between microbiological cure and treatment group. Other predictor variables of interest included sex, serum total protein, and age on the first day of diarrhea. Microbiological cure was defined as a negative fecal ELISA test at resolution of clinical diarrhea for calves with a positive ELISA test on the first day of diarrhea for at least one of the four fecal pathogens E. coli K99, bovine rotavirus and coronavirus, and C. parvum . Models were generated for each fecal pathogen individually and an overall model, which evaluated microbiological cure at clinical diarrhea resolution for calves that tested positive for any single pathogen on the first day of diarrhea. Serum total protein and calf age at first diarrhea were included in all final models to control for potential confounding by passive transfer status and age.", "Models were generated for each fecal pathogen individually and an overall model, which evaluated microbiological cure at clinical diarrhea resolution for calves that tested positive for any single pathogen on the first day of diarrhea. Serum total protein and calf age at first diarrhea were included in all final models to control for potential confounding by passive transfer status and age. Mean daily weight change. Linear regression was used to evaluate associations between ADG kg and treatment group during the treatment and pre-weaning periods separately. Other predictor variables of interest included sex, birth weight kg , serum total protein, number of days having diarrhea, age, and volume L of milk, Calva, and NuLife electrolytes fed at either end of treatment or hutch exit. For each calf, ADG during the treatment and pre-weaning period was calculated as the difference between birth and end treatment or hutch exit weight, respectively, divided by the number of days between these time points.", "Other predictor variables of interest included sex, birth weight kg , serum total protein, number of days having diarrhea, age, and volume L of milk, Calva, and NuLife electrolytes fed at either end of treatment or hutch exit. For each calf, ADG during the treatment and pre-weaning period was calculated as the difference between birth and end treatment or hutch exit weight, respectively, divided by the number of days between these time points. To explore the possibility of an interaction between treatment group, sex, and birth weight, the final linear regression model for ADG during the pre-weaning period was stratified by sex. Age at the end of treatment or hutch exit and number of days with diarrhea were dropped from all final models in favor of improved Akaike information criterion AIC . Onset of diarrhea and clinical cure. For all survival analyses, diarrhea was defined as a fecal score greater than 1 while diarrhea cure was defined as the second consecutive day of normal feces fecal score of 1 following the first diarrhea episode.", "Onset of diarrhea and clinical cure. For all survival analyses, diarrhea was defined as a fecal score greater than 1 while diarrhea cure was defined as the second consecutive day of normal feces fecal score of 1 following the first diarrhea episode. Subsequent episodes of diarrhea were not included in the analysis. Calves that died or did not experience diarrhea or cure from diarrhea were censored. If fresh feces were not observed on daily calf hutch assessment, a fecal score was not recorded for that day and not included in the analyses. Kaplan-Meier analysis was used to determine median days to first diarrhea event and, for those calves that developed diarrhea during the assessment period, median days to clinical diarrhea cure.", "If fresh feces were not observed on daily calf hutch assessment, a fecal score was not recorded for that day and not included in the analyses. Kaplan-Meier analysis was used to determine median days to first diarrhea event and, for those calves that developed diarrhea during the assessment period, median days to clinical diarrhea cure. A Log Rank test of equality was used to compare survivor functions between treatments. Cox Proportional Hazards regression analysis was used to estimate and compare the hazard of diarrhea and diarrhea cure between treatment groups. Sex, age, serum total protein at enrollment, birth weight kg , antimicrobial therapy, and application of fresh straw to the hutches were evaluated as predictor variables and potential confounders. When modeling the hazard of diarrhea cure, a binary variable termed therapeutic supplementation indicating whether calves were treated with either ZM, ZS or placebo for all or at least the first 5 days of diarrhea was evaluated as an additional covariate.", "Sex, age, serum total protein at enrollment, birth weight kg , antimicrobial therapy, and application of fresh straw to the hutches were evaluated as predictor variables and potential confounders. When modeling the hazard of diarrhea cure, a binary variable termed therapeutic supplementation indicating whether calves were treated with either ZM, ZS or placebo for all or at least the first 5 days of diarrhea was evaluated as an additional covariate. A five-day period was selected by the authors based on clinical experience, as five days represents a reasonable duration over which most therapeutic treatments for calf diarrhea should be applied and be expected to alleviate disease. The proportional hazards assumption that the hazard of diarrhea is independent of time was assessed using analysis of Schoenfeld residuals and testing whether the log hazard-ratio function is constant over time. Any variable found to violate the proportional hazards assumption was included in the final regression model as a time varying covariate. A total of 1,513 calves were enrolled in the trial.", "Any variable found to violate the proportional hazards assumption was included in the final regression model as a time varying covariate. A total of 1,513 calves were enrolled in the trial. However, due to failure to immediately recognize exclusion criteria, 23 calves were excluded shortly after enrollment. In addition, 8 calves were excluded due to treatment errors. Therefore, a total of 1,482 calves placebo = 500, ZM = 491, ZS = 491 were included in the final analyses. A total of 242 calves 16.3% had minimal fecal output at the time of enrollment while 125 calves 8.4% had abnormal fecal scores of 2 or 3 that were described as meconium.", "Therefore, a total of 1,482 calves placebo = 500, ZM = 491, ZS = 491 were included in the final analyses. A total of 242 calves 16.3% had minimal fecal output at the time of enrollment while 125 calves 8.4% had abnormal fecal scores of 2 or 3 that were described as meconium. All enrolled calves appeared healthy on visual assessment and hence were assumed to have a normal fecal score at the time of enrollment. The three treatment groups at enrollment did not differ significantly in mean birth weight kg P = 0.244 , mean serum total protein g/dL P = 0.541 , mean attitude score P = 0.845 , mean fecal score P = 0.522 , as shown in Table 1 , or distribution of calf sex P = 0.472 . Of the 1,482 study calves, 21 1.4% died during the trial: 5 calves in the placebo group 1.0% , 11 calves in the ZM group 2.2% , and 5 calves in the ZS group 1.0% . Of these 21 calves that died, 14 66.7% were bulls and 7 33.3% were heifers.", "Of the 1,482 study calves, 21 1.4% died during the trial: 5 calves in the placebo group 1.0% , 11 calves in the ZM group 2.2% , and 5 calves in the ZS group 1.0% . Of these 21 calves that died, 14 66.7% were bulls and 7 33.3% were heifers. Eighteen of the 21 calves 85.7% were found dead, rather than euthanized, due to acute and spontaneous death without previous obvious clinical signs of disease. The remaining 3 calves were euthanized prior to death due to severe and/or prolonged morbidity. Characteristics and causes of death based on field necropsy of these calves can be found in S4 Table. There was no significant difference in the proportion of calves that died between treatment groups P = 0.168 , though mortality was significantly higher in bulls compared to heifer calves P = 0.049 .", "Characteristics and causes of death based on field necropsy of these calves can be found in S4 Table. There was no significant difference in the proportion of calves that died between treatment groups P = 0.168 , though mortality was significantly higher in bulls compared to heifer calves P = 0.049 . Birth weight data were available for all 1,482 calves. Due to calf mortality between enrollment and completion of treatment n = 4 , end treatment weight data were available for 1,478 calves. Similarly, due to calf mortality n = 21 or missing data for body weight at hutch exit from the dairy's records n = 40 , hutch exit weight data were available for 1,421 calves. A summary of body weight data stratified by treatment group and sex is presented in Table 2 .", "Similarly, due to calf mortality n = 21 or missing data for body weight at hutch exit from the dairy's records n = 40 , hutch exit weight data were available for 1,421 calves. A summary of body weight data stratified by treatment group and sex is presented in Table 2 . Within each treatment group, bull calves showed consistently higher birth weight P < 0.001 , end treatment weight P < 0.001 , and exit hutch weight P < 0.001 compared to heifer calves. However, at birth, end of treatment, or hutch exit there were no differences in body weight between treatment groups for bulls P > 0.1 or heifers P > 0.1 . The mean attitude scores during the study period were 1.2 across the three treatment groups P = 0.208 . Of 1,482 calves included in the final analysis, A total of 629 treated milk samples were obtained throughout the 107-day study period and pooled by treatment group and week, yielding a total of 16 pooled samples per treatment group.", "The mean attitude scores during the study period were 1.2 across the three treatment groups P = 0.208 . Of 1,482 calves included in the final analysis, A total of 629 treated milk samples were obtained throughout the 107-day study period and pooled by treatment group and week, yielding a total of 16 pooled samples per treatment group. Zinc concentrations ppm were significantly higher in pooled milk samples treated with ZM P < 0.001 and ZS P < 0.001 compared to placebo-treated samples, and there were no significant differences between ZM and ZS-treated samples P = 1.000 , as shown in S5 Table. Within zinc treatment groups, oral zinc dose at the start and end of treatment is summarized by sex in S6 Table. For both ZM-and ZS-treated calves, oral zinc dose at the start P < 0.001 and end P < 0.001 of treatment was significantly higher in heifer versus bull calves. Serum zinc concentrations before and after treatment were obtained from 36 calves n = 12 for each treatment group and are summarized in S7 Table.", "For both ZM-and ZS-treated calves, oral zinc dose at the start P < 0.001 and end P < 0.001 of treatment was significantly higher in heifer versus bull calves. Serum zinc concentrations before and after treatment were obtained from 36 calves n = 12 for each treatment group and are summarized in S7 Table. Overall, there were no significant differences in mean pre-treatment serum zinc concentrations between treatment groups P = 0.233 . Mean post-treatment serum zinc concentrations were significantly higher in calves treated with ZM P < 0.001 and ZS P = 0.002 compared to placebo-treated calves, and there were no significant differences among calves treated with ZM and ZS P = 0.406 . Stratification of serum zinc data by treatment group and sex demonstrated that for ZM-treated calves, heifers had a numerically higher post-treatment serum zinc concentration compared to bulls, though the difference was not statistically significant P = 0.199 . In contrast, in ZStreated calves, heifers had a numerically lower post-treatment serum zinc concentration compared to bulls, though the difference was also not statistically significant P = 0.538 .", "Stratification of serum zinc data by treatment group and sex demonstrated that for ZM-treated calves, heifers had a numerically higher post-treatment serum zinc concentration compared to bulls, though the difference was not statistically significant P = 0.199 . In contrast, in ZStreated calves, heifers had a numerically lower post-treatment serum zinc concentration compared to bulls, though the difference was also not statistically significant P = 0.538 . Fecal analysis data were analyzed for 92 of the 127 randomly-selected calves. The remaining 35 calves were not included in the analysis due to not acquiring diarrhea during the assessment period n = 10 , death prior to final sampling n = 1 , exclusion due to improper treatment regimen n = 1 , or incorrect sampling day n = 23 .The 92 calves had a mean age at onset of diarrhea of 13.3, 11.0 and 11.3 days for ZM, ZS and placebo-treated groups, respectively; and a mean age at resolution of diarrhea of 18.8, 16.1 and 15.7 days for ZM, ZS and placebo-treated groups, respectively. There were no significant differences in the prevalence of E. coli K99 P = 0.694 , rotavirus P = 0.331 , coronavirus P = 0.819 , or C. parvum P = 0.719 fecal shedding on the first day of diarrhea between treatment groups S8 Table . There were no significant differences in the prevalence of E. coli K99 P = 0.256 , rotavirus P = 0.344 , or coronavirus P = 1.000 fecal shedding at resolution of diarrhea between treatment groups, though there was a difference in C. parvum P = 0.006 fecal shedding between treatment groups S9 Table .", "There were no significant differences in the prevalence of E. coli K99 P = 0.694 , rotavirus P = 0.331 , coronavirus P = 0.819 , or C. parvum P = 0.719 fecal shedding on the first day of diarrhea between treatment groups S8 Table . There were no significant differences in the prevalence of E. coli K99 P = 0.256 , rotavirus P = 0.344 , or coronavirus P = 1.000 fecal shedding at resolution of diarrhea between treatment groups, though there was a difference in C. parvum P = 0.006 fecal shedding between treatment groups S9 Table . The prevalence of C. parvum fecal shedding at resolution of diarrhea was significantly higher in calves treated with ZM P = 0.009 and ZS P = 0.023 compared to placebo-treated calves, and there were no significant differences among calves treated with ZM and ZS P = 1.000 . Results of logistic regression models for overall and pathogen-specific microbiological cure are presented in Tables 3-5 . For pathogen-specific cure, all calves that tested positive on the first day of diarrhea for coronavirus n = 4 tested negative at clinical diarrhea resolution. For calves that tested positive on the first day of diarrhea for E. coli K99 n = 9 , all placebo-treated calves n = 2 tested negative at clinical diarrhea resolution while half n = 2 of all ZS-treated calves tested either positive or negative at clinical diarrhea resolution, resulting in omission of ZS treatment variable from the model due to collinearity.", "For pathogen-specific cure, all calves that tested positive on the first day of diarrhea for coronavirus n = 4 tested negative at clinical diarrhea resolution. For calves that tested positive on the first day of diarrhea for E. coli K99 n = 9 , all placebo-treated calves n = 2 tested negative at clinical diarrhea resolution while half n = 2 of all ZS-treated calves tested either positive or negative at clinical diarrhea resolution, resulting in omission of ZS treatment variable from the model due to collinearity. Hence, logistic regression analyses for microbiological cure in calves that tested positive for coronavirus and E. coli K99 were not possible. For 59 calves that tested positive for rotavirus on the first day of diarrhea Table 3 , calves treated with ZM had a 50% increased odds of testing negative at diarrhea resolution compared to placebo-treated calves, though this difference was not significant P = 0.549 . Likewise, calves treated with ZS had 100% increased odds 2 times the odds of testing negative for rotavirus at diarrhea resolution compared to placebo-treated calves, though this difference was also not significant P = 0.314 . However, this model demonstrated a significant main effect of serum total protein, such that for every 1 unit g/dL increase in serum total protein at enrollment, the odds of microbiological cure of rotavirus decreased by 79% P = 0.026 .", "Likewise, calves treated with ZS had 100% increased odds 2 times the odds of testing negative for rotavirus at diarrhea resolution compared to placebo-treated calves, though this difference was also not significant P = 0.314 . However, this model demonstrated a significant main effect of serum total protein, such that for every 1 unit g/dL increase in serum total protein at enrollment, the odds of microbiological cure of rotavirus decreased by 79% P = 0.026 . For 40 calves that tested positive for Cryptosporidium parvum on the first day of diarrhea Table 4 , calves treated with ZM had an 87% reduced odds of testing negative at diarrhea resolution Effect of zinc on diarrhea in calves compared to placebo-treated calves, though this difference was not significant P = 0.119 . Likewise, calves treated with ZS had a 74% reduced odds of testing negative for Cryptosporidium parvum at diarrhea resolution compared to placebo-treated calves, though this difference was also not significant P = 0.183 . For 55 calves that tested positive for any one of the four fecal pathogens E. coli K99, rotavirus, coronavirus, Cryptosporidium parvum on the first day of diarrhea Table 5 , calves treated with ZM had a 25% increased odds of testing negative at diarrhea resolution compared to placebo-treated calves, though this difference was not significant P = 0.769 . Likewise, calves treated with ZS had a 52% increased odds of testing negative for Cryptosporidium parvum at diarrhea resolution compared to placebo-treated calves, though this difference was also not significant P = 0.633 .", "For 55 calves that tested positive for any one of the four fecal pathogens E. coli K99, rotavirus, coronavirus, Cryptosporidium parvum on the first day of diarrhea Table 5 , calves treated with ZM had a 25% increased odds of testing negative at diarrhea resolution compared to placebo-treated calves, though this difference was not significant P = 0.769 . Likewise, calves treated with ZS had a 52% increased odds of testing negative for Cryptosporidium parvum at diarrhea resolution compared to placebo-treated calves, though this difference was also not significant P = 0.633 . However, this model demonstrated that heifer calves had 71% lower odds of microbiological cure of any single fecal pathogen compared to bull calves P = 0.076 . A total of 1,482 calves were included in the linear regression model results for ADG during the treatment period, which are presented in Table 6 . There was no significant difference in ADG for ZM-or ZS-treated calves compared to placebo-treated calves, though there were significant main effects of sex, birth weight, and milk volume. Specifically, heifer calves gained 70 g bodyweight per day less compared to bull calves P < 0.001 .", "There was no significant difference in ADG for ZM-or ZS-treated calves compared to placebo-treated calves, though there were significant main effects of sex, birth weight, and milk volume. Specifically, heifer calves gained 70 g bodyweight per day less compared to bull calves P < 0.001 . For every 1 kg increase in birth weight, calves gained 16 g per day less than their herd mates P < 0.001 . For every 1 L increase in milk volume per day during the treatment period, calves gained an additional 13 g per day P < 0.001 . Table 7 summarizes the linear regression analysis of ADG during the pre-weaning period for 1,421 calves which showed a significant difference in ADG for ZM-treated calves compared to placebo-treated calves and in bull versus heifer calves. Milk volume had a significant effect on ADG, such that for every 1 L increase in milk volume per day during the treatment period, calves gained an additional 2 g bodyweight per day P = 0.001 .", "Table 7 summarizes the linear regression analysis of ADG during the pre-weaning period for 1,421 calves which showed a significant difference in ADG for ZM-treated calves compared to placebo-treated calves and in bull versus heifer calves. Milk volume had a significant effect on ADG, such that for every 1 L increase in milk volume per day during the treatment period, calves gained an additional 2 g bodyweight per day P = 0.001 . Results of the final model showed a significant main effect for ZM-treated bull calves and a significant interaction term for ZM treatment by sex. After controlling for milk volume received during the treatment Table 6 calves n = 1,482 Effect of zinc on diarrhea in calves period, ZM-treated bulls gained 22 g body weight per day on average more than placebotreated bull calves P = 0.042 and ZM-treated heifers gained 12 g less body weight per day on average compared to placebo-treated heifers P = 0.019 . When considering the model coefficients for treatment group, sex, and their interaction, bull calves treated with ZM gained 454 g per day 0.432 + 0.022 while female calves treated with ZM gained 0.404 g per day 0.432 + 0.022-0.016-0.034 , hence 50 g per day more gain in male calves compared to heifers treated with ZM P = 0.019 . For ZS-treated calves, there was a numerical decrease in weight gain of 5 g per day in bulls and 11 g per day in heifers compared to placebo-treated calves, though the differences were not significant P = 0.673 bulls; P = 0.681 heifers .", "When considering the model coefficients for treatment group, sex, and their interaction, bull calves treated with ZM gained 454 g per day 0.432 + 0.022 while female calves treated with ZM gained 0.404 g per day 0.432 + 0.022-0.016-0.034 , hence 50 g per day more gain in male calves compared to heifers treated with ZM P = 0.019 . For ZS-treated calves, there was a numerical decrease in weight gain of 5 g per day in bulls and 11 g per day in heifers compared to placebo-treated calves, though the differences were not significant P = 0.673 bulls; P = 0.681 heifers . Linear regression models of ADG during the pre-weaning period were stratified by sex in order to avoid interpreting a three-way interaction between treatment group, sex, and birth weight. In the heifer model S10 Table , the interaction between ZM treatment and birth weight was significant which implied that birth weight modified the effect of ZM treatment on ADG. At a 29 kg birth weight two standard deviations below the mean , ZM-treated heifers gained 49 g body weight per day on average less than placebo-treated heifers P = 0.037 . However, at a 49 kg birth weight two standard deviations above the mean , ZM-treated heifers gained 30 g body weight per day on average more than placebo-treated heifers P = 0.037 .", "At a 29 kg birth weight two standard deviations below the mean , ZM-treated heifers gained 49 g body weight per day on average less than placebo-treated heifers P = 0.037 . However, at a 49 kg birth weight two standard deviations above the mean , ZM-treated heifers gained 30 g body weight per day on average more than placebo-treated heifers P = 0.037 . In the bull calves model S11 Table there was no significant interaction between treatment group and birth weight. A total of 1,482 calves were included in the Kaplan-Meier survival analysis of time to first diarrhea event Fig 1 . There were no significant differences in median age at onset of diarrhea, specifically, 8, 8 and 7 days for the ZM, ZS and placebo-treated calves, respectively P = 0.402 . Cox proportional hazard regression model for diarrhea hazard are presented in Table 8 .", "There were no significant differences in median age at onset of diarrhea, specifically, 8, 8 and 7 days for the ZM, ZS and placebo-treated calves, respectively P = 0.402 . Cox proportional hazard regression model for diarrhea hazard are presented in Table 8 . After controlling for age, calves treated with ZM had a 14.7% reduced hazard of diarrhea compared to placebo-treated calves P = 0.015 . Calves treated with ZS had 13.9% reduced hazard of diarrhea compared to placebo-treated calves P = 0.022 . calves n = 1,421 A total of 1,394 calves were included in the Kaplan-Meier survival analysis of time to clinical diarrhea cure Fig 2 , as 88 calves failed to acquire diarrhea during the assessment period. There were no significant differences in the median days to diarrhea cure which was 7 days across all 3 treatment groups P = 0.264 .", "calves n = 1,421 A total of 1,394 calves were included in the Kaplan-Meier survival analysis of time to clinical diarrhea cure Fig 2 , as 88 calves failed to acquire diarrhea during the assessment period. There were no significant differences in the median days to diarrhea cure which was 7 days across all 3 treatment groups P = 0.264 . Cox proportional hazard regression model for diarrhea cure hazard are presented in Table 9 . Results of the final model showed a significant interaction term between treatment and therapeutic supplementation as well as the need for age as a time varying covariate. When considering calves that did not receive supplementation, respective to each of the 3 groups, for at least the first five days of diarrhea there was no significant difference between either ZM-and ZS-treated calves compared to placebo-treated calves P = 0.223 ZM, P = 0.134 ZS . However, when considering calves that were supplemented for at least the first five days of diarrhea, ZM-treated calves experienced a 21.4% higher hazard of cure from diarrhea compared to placebo-treated calves P = 0.027 .", "When considering calves that did not receive supplementation, respective to each of the 3 groups, for at least the first five days of diarrhea there was no significant difference between either ZM-and ZS-treated calves compared to placebo-treated calves P = 0.223 ZM, P = 0.134 ZS . However, when considering calves that were supplemented for at least the first five days of diarrhea, ZM-treated calves experienced a 21.4% higher hazard of cure from diarrhea compared to placebo-treated calves P = 0.027 . Likewise, ZS-treated calves experienced a 13.0% higher hazard of cure from diarrhea compared to placebo-treated calves P = 0.040 . The current trial demonstrated evidence for the beneficial effect of ZM on ADG and neonatal diarrhea as well as an effect of ZS on diarrhea in dairy calves during the pre-weaning period. It is important to consider these results in the context of the entire pre-weaning and hutch period. On average, after 90 days from birth to hutch exit, placebo-treated bull calves gained 38.88 kg body weight while ZM-treated bull calves gained an additional 1.98 kg 40.86 kg .", "It is important to consider these results in the context of the entire pre-weaning and hutch period. On average, after 90 days from birth to hutch exit, placebo-treated bull calves gained 38.88 kg body weight while ZM-treated bull calves gained an additional 1.98 kg 40.86 kg . In contrast, the effect of zinc on weight gain in treated heifers depended on birth weight. Low birth weight heifers treated with ZM gained on average less than a placebo-treated heifer of the same birth weight. In contrast, high birth weight heifers treated with ZM gained more than placebo-treated heifers of the same birth weight. The switch in direction of the association between ZM treatment and ADG in heifer calves depending on birth weight suggests a doseresponse effect rather than a true sex-specific effect of ZM on ADG.", "In contrast, high birth weight heifers treated with ZM gained more than placebo-treated heifers of the same birth weight. The switch in direction of the association between ZM treatment and ADG in heifer calves depending on birth weight suggests a doseresponse effect rather than a true sex-specific effect of ZM on ADG. Hence, low birth weight calves including heifers may require a lower dose of ZM to mitigate any negative effect of what is otherwise a suitable dose for higher birth weight calves. These findings are in agreement with a previous randomized clinical trial testing the effect of daily oral zinc in diarrheic neonatal Holstein calves which, showed that ZM-treated calves had a numerically, though not significantly increased ADG compared to calves treated with zinc oxide or placebo due to small sample size . In general, our trial findings are in agreement with the large body of human literature supporting the use of oral zinc for the prevention and treatment of diarrhea and impaired growth in children . Zinc supplementation is widely accepted by global health organizations as a vital component of therapy for childhood diarrhea , however, recent reviews of the literature demonstrated heterogeneity in study results on the basis of age, baseline zinc status, geographic location, and supplementation regimen .", "In general, our trial findings are in agreement with the large body of human literature supporting the use of oral zinc for the prevention and treatment of diarrhea and impaired growth in children . Zinc supplementation is widely accepted by global health organizations as a vital component of therapy for childhood diarrhea , however, recent reviews of the literature demonstrated heterogeneity in study results on the basis of age, baseline zinc status, geographic location, and supplementation regimen . Similar to our findings, a sex-specific response to zinc supplementation has been demonstrated in several human studies. Zinc gluconate administered for diarrhea prevention reduced the incidence of dysentery in treated boys but not girls ; when given therapeutically, it reduced diarrhea duration and frequency more dramatically in boys compared to girls . Similarly, zinc sulfate was shown to improve Effect of zinc on diarrhea in calves diarrhea outcomes in boys but improved growth rates in girls . Broadly, these differences between male and female responses to zinc supplementation are not understood, though theories regarding differences in immune function and response , diarrhea etiology , and nutrient requirements have been proposed.", "Similarly, zinc sulfate was shown to improve Effect of zinc on diarrhea in calves diarrhea outcomes in boys but improved growth rates in girls . Broadly, these differences between male and female responses to zinc supplementation are not understood, though theories regarding differences in immune function and response , diarrhea etiology , and nutrient requirements have been proposed. In the current study, ZM-treated bulls demonstrated increased ADG compared to placebo-treated bulls while ZM-treated heifers demonstrated decreased ADG compared to placebo-treated heifers. However, due to a significant interaction between ZM treatment and birth weight, this reduction in ADG in ZMtreated heifers was overcome with increasing birth weight, such that ZM-treated heifers with birth weights above 42 kg experienced increased ADG during the pre-weaning period, compared to placebo-treated heifers with birth weights above 42 kg. Differences in the growth response to ZM supplementation between bull and heifer calves may have been related to its effect on feed intake. Previous research on the effects of feeding various doses of oral zinc oxide to pre-ruminant dairy calves demonstrated that high levels of oral zinc supplementation resulted in reduced feed intake .", "Differences in the growth response to ZM supplementation between bull and heifer calves may have been related to its effect on feed intake. Previous research on the effects of feeding various doses of oral zinc oxide to pre-ruminant dairy calves demonstrated that high levels of oral zinc supplementation resulted in reduced feed intake . In the current trial, oral ZM dose was estimated to be significantly higher in heifers compared to bulls due to the significantly lower birth weight of heifers. Additionally, serum zinc concentrations in ZM-treated heifers were numerically higher than that of bulls, though this difference was not significant, likely due to the small sample size. Perhaps the higher zinc dose in heifers was associated with reduced feed intake, leading to reduced growth, and that this effect was more pronounced for ZM compared to ZS. The fact that ZM-treated heifers with birth weights approaching those of average bull calves and, therefore, a similar zinc dose to that in bulls experienced an increase in ADG over placebo-treated heifers similar to that of bull calves partially supports this theory.", "Perhaps the higher zinc dose in heifers was associated with reduced feed intake, leading to reduced growth, and that this effect was more pronounced for ZM compared to ZS. The fact that ZM-treated heifers with birth weights approaching those of average bull calves and, therefore, a similar zinc dose to that in bulls experienced an increase in ADG over placebo-treated heifers similar to that of bull calves partially supports this theory. Although management practices on the study dairy were designed to be identical for both bulls and heifers, it is possible that subtle, unrecognized differences in nutritional and health management may also have contributed to sex-specific differences in weight gain. Nevertheless, future trials are warranted to investigate the potential differences in the dose-response to zinc supplementation between bulls and heifers. We hypothesized that ADG would be increased in zinc-supplemented calves compared to placebo-supplemented calves due to the potential preventive and therapeutic effects of zinc supplementation on neonatal diarrhea. In other words, calf diarrhea is mitigated by zinc supplementation and, therefore, on the causal pathway between zinc and ADG.", "We hypothesized that ADG would be increased in zinc-supplemented calves compared to placebo-supplemented calves due to the potential preventive and therapeutic effects of zinc supplementation on neonatal diarrhea. In other words, calf diarrhea is mitigated by zinc supplementation and, therefore, on the causal pathway between zinc and ADG. However, considering the similarly-reduced hazard of diarrhea and increased hazard of cure from diarrhea in both ZM and ZS treatment groups but a lack of effect of ZS on ADG, it is likely that the effect of ZM on ADG is not solely mediated through its effects on diarrhea. Differences in effectiveness between organic and inorganic formulations also may exist. In fact, the underlying mechanism of action of oral zinc remains unknown . Several theories of the mechanisms of action of zinc in the prevention and treatment of childhood diarrhea exist, including a mucosal-protective role, a diarrhea-induced zinc deficiency, an essential element in cell-mediated immunity, and a modifier of intra-luminal electrolyte secretion and absorption 6, .", "In fact, the underlying mechanism of action of oral zinc remains unknown . Several theories of the mechanisms of action of zinc in the prevention and treatment of childhood diarrhea exist, including a mucosal-protective role, a diarrhea-induced zinc deficiency, an essential element in cell-mediated immunity, and a modifier of intra-luminal electrolyte secretion and absorption 6, . The clinical and practical implications of effects of ZM supplementation on ADG and diarrhea must be considered. Pre-weaned calf diarrhea remains an ongoing issue for the dairy industry. The deleterious effects on calf health and performance and the resulting economic burden create a strong incentive to treat and prevent diarrhea in pre-weaned calves. On large dairy operations like those in California's Central Valley, small changes in disease incidence and duration as well as animal growth and performance can have profound economic consequences.", "The deleterious effects on calf health and performance and the resulting economic burden create a strong incentive to treat and prevent diarrhea in pre-weaned calves. On large dairy operations like those in California's Central Valley, small changes in disease incidence and duration as well as animal growth and performance can have profound economic consequences. As a non-antimicrobial product, zinc may become increasingly attractive as antimicrobials in livestock feed are under increased scrutiny and regulation due to concerns about antimicrobial resistance . Prevalence of C. parvum fecal shedding in a random sample of 92 study calves at onset and resolution of diarrhea was significantly higher in calves treated with zinc compared to Placebo-treated calves. In contrast, a previous study where calves that tested positive for C. parvum at the start of diarrhea and were treated with ZM had 16 times higher odds of being fecal ELISA negative at exit compared to the Placebo group P = 0.08; power = 72.3% . The difference in findings may be due to the differences in the timing of diarrhea across treatment groups.", "In contrast, a previous study where calves that tested positive for C. parvum at the start of diarrhea and were treated with ZM had 16 times higher odds of being fecal ELISA negative at exit compared to the Placebo group P = 0.08; power = 72.3% . The difference in findings may be due to the differences in the timing of diarrhea across treatment groups. For the current study's random sample of calves that acquired, survived, and were sampled on the correct days, the mean age of calves on both onset and resolution of diarrhea was higher for ZM and ZS calves compared to placebo-treated calves. Although C. parvum oocyst shedding in infected calves can occur as early as 3 days of age, peak shedding occurs at about 14 days of age . It is possible that the increase in prevalence of C. parvum shedding in ZM and ZS treated groups was due to the increased age of zinc-treated calves compared to placebo-treated calves at resolution of diarrhea. The latter explanation is also supported by our findings that the odds of microbiological cure from C. parvum significantly decreased in older calves, with no significant differences in the odds of cure between treatment groups.", "It is possible that the increase in prevalence of C. parvum shedding in ZM and ZS treated groups was due to the increased age of zinc-treated calves compared to placebo-treated calves at resolution of diarrhea. The latter explanation is also supported by our findings that the odds of microbiological cure from C. parvum significantly decreased in older calves, with no significant differences in the odds of cure between treatment groups. In addition, the current testing did not estimate the concentration of C. parvum shedding which may still differ between treatment groups. Despite the large sample size, the current trial was limited to a single California dairy, which may represent other large dairies but does not reflect all the dairy management systems in California or elsewhere. Additionally, our results show that calves respond to zinc supplementation for diarrhea prevention differently depending on chemical formulation and calf sex. The latter could be due to differences in body weight between bulls and heifers and may point towards the need for sex-specific dosing.", "Additionally, our results show that calves respond to zinc supplementation for diarrhea prevention differently depending on chemical formulation and calf sex. The latter could be due to differences in body weight between bulls and heifers and may point towards the need for sex-specific dosing. Furthermore, the current research did not evaluate the potential economic utility of zinc supplementation. Future studies on more accurate dosing of zinc by calf sex, the practical feasibility of weight-based dosing, and the expected cost-effectiveness of zinc administration as part of the management of pre-weaned dairy calves are warranted. Finally, our clinical trial was performed on a single, large, predominately Holstein, California dairy over a six-month period, which precluded our ability to evaluate differences due to season or breed. Hence, future studies to assess any modifying effect of breed and seasonal differences on the effect of zinc on calf health and weight gain are also needed.", "Finally, our clinical trial was performed on a single, large, predominately Holstein, California dairy over a six-month period, which precluded our ability to evaluate differences due to season or breed. Hence, future studies to assess any modifying effect of breed and seasonal differences on the effect of zinc on calf health and weight gain are also needed. The current double blind, block-randomized placebo controlled clinical trial tested the effect of a prophylactic daily oral zinc supplementation in neonatal Holstein calves. Bull calves treated with ZM had a significantly increased ADG 22 g per day during the pre-weaning period compared to placebo-treated bulls. In comparison, ZM-treated heifers had significantly lower average daily gain 9 g per day compared to placebo-treated heifers, although higher ZM doses in low birthweight heifers may explain the lower ADG. Calves treated with either ZM or ZS had significantly lower risks of diarrhea and significantly higher risk of cure from diarrhea over the first 30 days of life compared to placebo-treated calves and hence the current trial demonstrated that zinc supplementation delayed diarrhea and expedited diarrhea recovery in pre-weaned calves.", "In comparison, ZM-treated heifers had significantly lower average daily gain 9 g per day compared to placebo-treated heifers, although higher ZM doses in low birthweight heifers may explain the lower ADG. Calves treated with either ZM or ZS had significantly lower risks of diarrhea and significantly higher risk of cure from diarrhea over the first 30 days of life compared to placebo-treated calves and hence the current trial demonstrated that zinc supplementation delayed diarrhea and expedited diarrhea recovery in pre-weaned calves. Additionally, zinc improved weight gain differentially in bulls compared to heifers, indicating the need for further research to investigate zinc dosing in calves. Supporting information S1 DOCX S1 Dataset. Raw data collected from trial, organized as separate excel sheets for enrollment, daily assessment, serum total protein, birth weight, exit treatment weight, exit trial weight, serum zinc testing, fecal samples, fecal testing, milk testing, and dead calves. XLSX" ]

[ "The objective of this clinical trial was to evaluate the effectiveness of zinc supplementation on diarrhea and average daily weight gain ADG in pre-weaned dairy calves. A total of 1,482 healthy Holstein heifer and bull calves from a large California dairy were enrolled at 24 to 48 hours of age until hutch exit at approximately 90 days of age. Calves were block-randomized by time to one of three treatments: 1 placebo, 2 zinc methionine ZM , or 3 zinc sulfate ZS administered in milk once daily for 14 days. Serum total protein at enrollment and body weight at birth, treatment end, and hutch exit were measured. Fecal consistency was assessed daily for 28 days post-enrollment. For a random sample of 127 calves, serum zinc concentrations before and after treatment and a fecal antigen ELISA at diarrhea start and resolution for Escherichia coli K99, rotavirus, coronavirus, and Cryptosporidium parvum were performed.", "Fecal consistency was assessed daily for 28 days post-enrollment. For a random sample of 127 calves, serum zinc concentrations before and after treatment and a fecal antigen ELISA at diarrhea start and resolution for Escherichia coli K99, rotavirus, coronavirus, and Cryptosporidium parvum were performed. Linear regression showed that ZM-treated bull calves had 22 g increased ADG compared to placebo-treated bulls P = 0.042 . ZM-treated heifers had 9 g decreased ADG compared to placebo-treated heifers P = 0.037 , after adjusting for average birth weight. Sex-stratified models showed that high birth weight heifers treated with ZM gained more than placebo-treated heifers of the same birth weight, which suggests a dose-response effect rather than a true sex-specific effect of ZM on ADG. Cox regression showed that ZM and ZS-treated calves had a 14.7% P = 0.015 and 13.9% P = 0.022 reduced hazard of diarrhea, respectively, compared to placebo-treated calves.", "Sex-stratified models showed that high birth weight heifers treated with ZM gained more than placebo-treated heifers of the same birth weight, which suggests a dose-response effect rather than a true sex-specific effect of ZM on ADG. Cox regression showed that ZM and ZS-treated calves had a 14.7% P = 0.015 and 13.9% P = 0.022 reduced hazard of diarrhea, respectively, compared to placebo-treated calves. Calves supplemented for at least the first five days of diarrhea with ZM and ZS had a 21.4% P = 0.027 and 13.0% P = 0.040 increased hazard of cure from diarrhea, respectively, compared to placebo-treated calves. Logistic regression showed that the odds of microbiological cure at diarrhea resolution for rotavirus, C. parvum, or any single fecal pathogen was not different between treatment groups. Zinc supplementation delayed diarrhea and expedited diarrhea recovery in pre-weaned calves. Additionally, zinc improved weight gain differentially in bulls compared to heifers, indicating a research need for sex-specific dosing.", "Zinc supplementation delayed diarrhea and expedited diarrhea recovery in pre-weaned calves. Additionally, zinc improved weight gain differentially in bulls compared to heifers, indicating a research need for sex-specific dosing. Text: Introduction Diarrhea is the leading cause of morbidity and mortality and the most common reason for antimicrobial drug treatments in pre-weaned dairy heifers . A USDA survey of preweaned dairy heifers reported that 24% experienced diarrhea and 18% received antimicrobial treatment for it . Diarrhea is also a leading cause of morbidity and the second foremost cause of mortality in children with over 1 billion cases and a half a million deaths annually . Zinc supplementation in children decreases the incidence, duration, and severity of diarrhea, increases recovery rates, decreases the use of antibiotics and antidiarrheal medications, and reduces mortality .", "Diarrhea is also a leading cause of morbidity and the second foremost cause of mortality in children with over 1 billion cases and a half a million deaths annually . Zinc supplementation in children decreases the incidence, duration, and severity of diarrhea, increases recovery rates, decreases the use of antibiotics and antidiarrheal medications, and reduces mortality . In a clinical trial that established a non-toxic zinc dose and investigated its therapeutic use for diarrhea in neonatal dairy calves, zinc-treated calves had numerically quicker clinical recovery, increased weight gain, and higher odds of fecal clearance of Cryptosporidium parvum between diarrhea onset and recovery compared with placebotreated calves . As a result, zinc supplementation may be beneficial for prevention of diarrhea in dairy calves and, thus, minimize antimicrobial use. However, studies investigating zinc's potential effectiveness are lacking. In children, both organic zinc acetate, zinc gluconate, and zinc methionine and inorganic zinc sulfate and zinc oxide zinc formulations are beneficial in the prevention and treatment of childhood diarrhea .", "However, studies investigating zinc's potential effectiveness are lacking. In children, both organic zinc acetate, zinc gluconate, and zinc methionine and inorganic zinc sulfate and zinc oxide zinc formulations are beneficial in the prevention and treatment of childhood diarrhea . However, differing bioavailability was observed in several animal studies . In addition, the underlying mechanism of action of oral zinc is unknown . Hence, contrasting the effect, if any, of organic compared to inorganic zinc formulations in pre-weaned calves may help identify differences in mode of action. The objective of this clinical trial was to compare average daily weight gain ADG and the incidence and duration of diarrhea in pre-weaned dairy calves randomly assigned to receive either organic zinc methionine ZM , inorganic zinc sulfate ZS , or a placebo in milk once daily for 14 days.", "Hence, contrasting the effect, if any, of organic compared to inorganic zinc formulations in pre-weaned calves may help identify differences in mode of action. The objective of this clinical trial was to compare average daily weight gain ADG and the incidence and duration of diarrhea in pre-weaned dairy calves randomly assigned to receive either organic zinc methionine ZM , inorganic zinc sulfate ZS , or a placebo in milk once daily for 14 days. By elucidating the potential role of zinc supplementation in prevention of diarrhea in preweaned dairy calves, calf morbidity, mortality, and antimicrobial usage may be mitigated. A double-blind, block randomized, placebo-controlled clinical trial was conducted between December 14, 2015 and June 15, 2016 on a large dairy in California's San Joaquin Valley. The dairy was selected based on the owner and calf manager's willingness to participate in the study. The dairy herd was composed of 5,500 lactating cows, predominantly Holsteins, and housed approximately 1,600 pre-weaned calves.", "The dairy was selected based on the owner and calf manager's willingness to participate in the study. The dairy herd was composed of 5,500 lactating cows, predominantly Holsteins, and housed approximately 1,600 pre-weaned calves. Approximately 75% of the calves were born on the participating dairy and 25% were born on an affiliated dairy located approximately 10 miles away. Calves enrolled in the trial included healthy Holstein heifer or bull calves 24 to 48 hours of age. Calves were determined to be healthy via visual examination by a veterinarian HF or a trained researcher. Calves were excluded if they had obvious morbidities or congenital defects, were non-Holstein, born on the affiliated dairy, younger than 24 hours of age or older than 48 hours of age at the time of enrollment.", "Calves were determined to be healthy via visual examination by a veterinarian HF or a trained researcher. Calves were excluded if they had obvious morbidities or congenital defects, were non-Holstein, born on the affiliated dairy, younger than 24 hours of age or older than 48 hours of age at the time of enrollment. Calves from the affiliated dairy were excluded due to differences in physical location and management practices of pre-partum cows. All procedures were approved by the University of California Davis Institutional Animal Care and Use Committee protocol number 18067 Approved: March 6, 2014 . 107 g between treatment groups Stata, College Station, TX . After allowing for 15% attrition and assuming 50% incidence of diarrhea based on study authors expert opinion, and a difference in ADG of 107g , a sample size of 500 calves per treatment group n = 1500 total was deemed required.", "107 g between treatment groups Stata, College Station, TX . After allowing for 15% attrition and assuming 50% incidence of diarrhea based on study authors expert opinion, and a difference in ADG of 107g , a sample size of 500 calves per treatment group n = 1500 total was deemed required. Newborn calves were removed from the dam within an hour of birth and placed in a strawbedded, group calf pen where their navels were dipped in an iodine-based solution. Each calf received 4 liters of colostrum within 1 hour of birth and a second colostrum feeding 2 liters 6-10 hours after birth. Colostrum was refrigerated for < 48 hours and heated in a hot water bath prior to feeding using an esophageal tube feeder. Within 18 hours of birth, pre-weaned calves were transported to individual metal hutches initially bedded with almond shells.", "Colostrum was refrigerated for < 48 hours and heated in a hot water bath prior to feeding using an esophageal tube feeder. Within 18 hours of birth, pre-weaned calves were transported to individual metal hutches initially bedded with almond shells. Straw hay was later added to wet and muddy hutches throughout the pre-weaning period. For the first 14 days of life, pre-weaned calves were bottle-fed 1.9 liters of milk twice daily and 1.9 liters of a commercial oral electrolyte solution Calva Lyte; Calva Products, Inc., Acampo, CA once daily between milk feedings. Milk consisted of a combination of pasteurized waste milk, rehydrated commercial milk replacer powder Strauss Feeds LLC, Watertown, WI , tetracycline and neomycin powder, and additional supplements S1 Table . The proportion of pasteurized waste milk to milk replacer varied with each feeding, as the volume of waste milk varied with changes in the number and production of cows contributing to the waste milk tank.", "Milk consisted of a combination of pasteurized waste milk, rehydrated commercial milk replacer powder Strauss Feeds LLC, Watertown, WI , tetracycline and neomycin powder, and additional supplements S1 Table . The proportion of pasteurized waste milk to milk replacer varied with each feeding, as the volume of waste milk varied with changes in the number and production of cows contributing to the waste milk tank. After 14 days of age, calves were bottle-fed 2.8 liters of milk twice daily. Calves with clinical diarrhea received 1.9 liters of a commercial oral electrolyte solution NuLife; Genex Cooperative, Inc., Shawano, WI once daily between milk feedings. A list of ingredients that make up the two oral electrolyte solutions can be found in S2 Table. All pre-weaned calves had free choice access to water and a calf starter grain mix.", "A list of ingredients that make up the two oral electrolyte solutions can be found in S2 Table. All pre-weaned calves had free choice access to water and a calf starter grain mix. Calves were gradually weaned over a 10-day period, starting at approximately 60 days of age after which calves received a grower grain mix until 90 days of age when they were moved to group pens. Each calf received 1 mL of a selenium supplement MU-SE; Merck Animal Health, Boxmeer, Netherlands intramuscularly within 24 hours of age and an intranasal vaccine Inforce 3, Zoetis, Inc., Florham Park, NJ within 48 hours of age and again near the time of weaning. Approximately 8.3% n = 126 of all enrolled calves were also vaccinated using an autogenous Moraxella bovis/bovoculi bacterin vaccine Newport Laboratories, Inc., Worthington, MN for the prevention of pinkeye at 5 and 7 weeks of age. All pre-weaned calves were evaluated daily by dairy personnel for calfhood diseases and treated according to standard on-farm treatment protocols.", "Approximately 8.3% n = 126 of all enrolled calves were also vaccinated using an autogenous Moraxella bovis/bovoculi bacterin vaccine Newport Laboratories, Inc., Worthington, MN for the prevention of pinkeye at 5 and 7 weeks of age. All pre-weaned calves were evaluated daily by dairy personnel for calfhood diseases and treated according to standard on-farm treatment protocols. With regard to diarrhea therapy, calves less than two weeks of age with clinical diarrhea received an oral mixture of 118.5 mL 2.08 g bismuth subsalicylate Bismusal Suspension, Durvet, Inc., Blue Springs, MO and 31.5 mL 1575 mg spectinomycin SpectoGard, Bimeda, Inc., Le Sueur, MN once daily for two days. Calves older than two weeks of age with clinical diarrhea received oral sulfamethoxazole 1600 mg /trimethoprim 320 mg Amneal Pharmaceuticls of NY, Hauppauge, NY once daily for 2 to 3 days. Repeated treatment was at the discretion of the calf manager. inductively coupled plasma mass spectrometry .", "Repeated treatment was at the discretion of the calf manager. inductively coupled plasma mass spectrometry . Financial limitations restricted the ability to test more than two samples of each dietary component. Due to variation of zinc content in duplicate samples, the maximum concentration was used to estimate the daily zinc intake during the first 14 days of life S3 Table . In blocks of 36, enrolled calves were randomized using a random number generator Microsoft Corporation, Redmond, WA to one of three treatment groups: 1 placebo, 2 ZM, or 3 ZS to be administered in the morning milk feeding once daily for 14 days, starting on the day after enrollment. During the 14-day zinc treatment period, study calves that did not drink the entire milk bottle were tube-fed the remaining milk by trained technicians using an esophageal feeder disinfected between uses.", "In blocks of 36, enrolled calves were randomized using a random number generator Microsoft Corporation, Redmond, WA to one of three treatment groups: 1 placebo, 2 ZM, or 3 ZS to be administered in the morning milk feeding once daily for 14 days, starting on the day after enrollment. During the 14-day zinc treatment period, study calves that did not drink the entire milk bottle were tube-fed the remaining milk by trained technicians using an esophageal feeder disinfected between uses. The ZM treatment group received 0.45 g zinc methionine complex equivalent to 80 mg of elemental zinc as the product Zinpro180 Zinpro Corporation, Eden Prairie, MN combined with 0.44 g milk replacer powder. The ZS treatment group received 0.22 g zinc sulfate monohydrate equivalent to 80 mg of elemental zinc Sigma-Aldrich Company, St. Louis, MO combined with 0.44 g milk replacer powder. The placebo treatment group received approximately 0.44 g fresh milk replacer powder. Zinc supplementation was based on a previously published clinical trial, toxicological studies, and nutritional guidelines 11, .", "The placebo treatment group received approximately 0.44 g fresh milk replacer powder. Zinc supplementation was based on a previously published clinical trial, toxicological studies, and nutritional guidelines 11, . The milk replacer powder used in treatment preparation was the same product used in the pre-weaned calf milk ration. Treatments were weighed GX-2000 precision scale; A&D Co Ltd., San Jose, CA at the Dairy Epidemiology Laboratory at the University of California, Davis Veterinary Medicine Teaching and Research Center VMTRC; Aly Lab in Tulare, CA and placed in 2.0 mL microcentrifuge tubes with polypropylene snap caps Fisher Scientific, Pittsburgh, PA . Prior to study commencement, a color was randomly and permanently assigned to each treatment group, after which treatment tubes, calf milk bottles and calf hutches were marked with either pink, orange, or yellow ink. The study investigators and technicians responsible for treatment preparation, allocation, administration and data collection were blinded to the color assignment until completion of the trial.", "Prior to study commencement, a color was randomly and permanently assigned to each treatment group, after which treatment tubes, calf milk bottles and calf hutches were marked with either pink, orange, or yellow ink. The study investigators and technicians responsible for treatment preparation, allocation, administration and data collection were blinded to the color assignment until completion of the trial. For each calf, the study period started at enrollment 24 to 48 hours of age and ended when the calf exited the hutch approximately 90 days of age and from here onwards will be referred to as the \"pre-weaning period.\" Calf enrollment and study procedures were performed daily at the time of morning feeding. At enrollment, calf characteristics, including sex, birth date, time of first colostrum feeding, and treatment color were recorded. Attitude and feces were assessed daily until 28 days post-enrollment using previously published methods by two study investigators, a veterinarian and a trained researcher.", "At enrollment, calf characteristics, including sex, birth date, time of first colostrum feeding, and treatment color were recorded. Attitude and feces were assessed daily until 28 days post-enrollment using previously published methods by two study investigators, a veterinarian and a trained researcher. Attitude scoring was based on a threepoint scale. A calf with an attitude score of 1 was bright, alert, and readily stood with stimulation; a calf with a score of 2 was quiet, alert, and stood only with moderate stimulation; a calf with a score of 3 exhibited a dull mentation and remained recumbent in response to stimulation. Fecal scoring was performed only on fresh feces and was also based on a three-point scale, as 1 solid , 2 semi-formed/loose , or 3 watery . If no fresh feces were observed in the hutch, \"none seen\" NS was recorded.", "Fecal scoring was performed only on fresh feces and was also based on a three-point scale, as 1 solid , 2 semi-formed/loose , or 3 watery . If no fresh feces were observed in the hutch, \"none seen\" NS was recorded. Body weight was measured using a digital scale at birth, end of treatment, and hutch exit by farm employees with the exception of end of treatment weights, which were recorded by study investigators. Treatments for farm-diagnosed illnesses were performed and recorded on hutch cards by the calf manager. Study investigators regularly recorded this information from cards in addition to extracting treatment event reports from DairyComp 305 Valley Agricultural Software, Tulare, CA . Though daily diagnosis and treatment of study calves was performed and recorded by the calf manager, a veterinarian was responsible for examining and determining whether study calves met specific criteria for euthanasia.", "Study investigators regularly recorded this information from cards in addition to extracting treatment event reports from DairyComp 305 Valley Agricultural Software, Tulare, CA . Though daily diagnosis and treatment of study calves was performed and recorded by the calf manager, a veterinarian was responsible for examining and determining whether study calves met specific criteria for euthanasia. A calf was euthanized if morbidity was severe enough to significantly depress appetite, hydration status, attitude, mentation, and/or ambulatory capability and the calf showed limited to no immediate response to therapy or supportive care. Study calves were euthanized by the calf manager using an on-farm captive bolt protocol established by the herd veterinarian within 3 hours of the decision to euthanize. Enrolled calves that died prior to hutch exit were necropsied within 24 hours of death by a veterinarian. All calves were monitored throughout the study period for evidence of zinc toxicity.", "Enrolled calves that died prior to hutch exit were necropsied within 24 hours of death by a veterinarian. All calves were monitored throughout the study period for evidence of zinc toxicity. At the end of the study period, calves were cared for at the dairy in accordance with standard commercial operations. Using the same random number generator, a random sample of 127 calves was selected for additional biologic sampling. Approximately 8 to 10% of the study population was selected due to the financial constraints of additional laboratory testing. Serum zinc concentration at baseline and in response to treatment were evaluated for the three treatment groups.", "Approximately 8 to 10% of the study population was selected due to the financial constraints of additional laboratory testing. Serum zinc concentration at baseline and in response to treatment were evaluated for the three treatment groups. Feces collected on the first day of diarrhea and at diarrhea resolution were evaluated for four fecal pathogens Escherichia. coli K99, bovine rotavirus and coronavirus, and Cryptosporidium parvum oocysts . Serum total protein. At enrollment, blood from each calf was collected from the jugular vein using a 20 gauge 1-inch multi-sample needle Exelint International Co., Redondo Beach, CA and placed into a 10 mL red top serum tube BD Vacutainer, Franklin Lakes, NJ for determination of total protein.", "Serum total protein. At enrollment, blood from each calf was collected from the jugular vein using a 20 gauge 1-inch multi-sample needle Exelint International Co., Redondo Beach, CA and placed into a 10 mL red top serum tube BD Vacutainer, Franklin Lakes, NJ for determination of total protein. Samples remained at room temperature for up to 12 hours until clotting and were then centrifuged International Equipment Company, CRU-5000, Needham Heights, MA for 15 minutes. Total protein g/dL was measured by a single investigator HF on decanted serum using a handheld refractometer Sper Scientific, Model 300005, Scottsdale, AZ . Serum zinc. For the 127 randomly-sampled calves, additional blood was collected at enrollment and on the last day of treatment, as described above, and placed into 6.0 mL trace element tubes BD Vacutainer, Franklin Lakes, NJ .", "Serum zinc. For the 127 randomly-sampled calves, additional blood was collected at enrollment and on the last day of treatment, as described above, and placed into 6.0 mL trace element tubes BD Vacutainer, Franklin Lakes, NJ . Serum was extracted, as described above, and placed in a 2.0 mL microcentrifuge tube with a polypropylene snap cap Fisher Scientific, Pittsburgh, PA and stored at −20˚C until analysis. Using the same random number generator, 36 of the 127 sampled calves were randomly selected for analysis due to limited financial resources. The pre-and post-zinc supplementation serum samples from each calf n = 72 were analyzed for zinc concentration ppm by ICP-OES inductively coupled plasma-optical emission spectroscopy at the CAHFS Laboratory. Quality control samples, including method blanks, laboratory control spikes, and reference Sigma serum, were run with each set of study samples.", "The pre-and post-zinc supplementation serum samples from each calf n = 72 were analyzed for zinc concentration ppm by ICP-OES inductively coupled plasma-optical emission spectroscopy at the CAHFS Laboratory. Quality control samples, including method blanks, laboratory control spikes, and reference Sigma serum, were run with each set of study samples. Fecal analysis. For 127 randomly-sampled calves at the first diarrhea episode, fecal samples were collected at two time points, the first day of diarrhea fecal score > 1 and the day diarrhea resolved second day of fecal score = 1 . Using new gloves and sterile lubricant, fresh feces was collected by digital rectal stimulation into 20 mL polypropylene twist-top jars The Cary Company, Addison, IL and stored at -20˚C until analysis. Fecal samples were tested at the Dairy Epidemiology Laboratory, VMTRC by a veterinarian for E. coli K99, bovine rotavirus and coronavirus, and C. parvum oocysts using a commercial kit Pathasure Enteritis 4; Biovet, Quebec, Canada that is highly specific > 90% and sensitive E. coli K99, 93%; rotavirus, 100%; coronavirus, 77% .", "Using new gloves and sterile lubricant, fresh feces was collected by digital rectal stimulation into 20 mL polypropylene twist-top jars The Cary Company, Addison, IL and stored at -20˚C until analysis. Fecal samples were tested at the Dairy Epidemiology Laboratory, VMTRC by a veterinarian for E. coli K99, bovine rotavirus and coronavirus, and C. parvum oocysts using a commercial kit Pathasure Enteritis 4; Biovet, Quebec, Canada that is highly specific > 90% and sensitive E. coli K99, 93%; rotavirus, 100%; coronavirus, 77% . For calves with a first-day diarrhea sample on or before 7 days of age, both fecal samples were tested for all four pathogens. For calves with a first-day diarrhea sample after 7 days of age, both fecal samples were tested for three pathogens C. parvum, bovine rotavirus and coronavirus . Samples from calves older than 7 days of age were not tested for E. coli K99 based on calves' susceptibility . Testing was performed according to kit manufacturer guidelines, and a low-temperature incubator Fisher Scientific, Model 146, Pittsburgh, PA was used during incubation periods.", "Samples from calves older than 7 days of age were not tested for E. coli K99 based on calves' susceptibility . Testing was performed according to kit manufacturer guidelines, and a low-temperature incubator Fisher Scientific, Model 146, Pittsburgh, PA was used during incubation periods. Test results were recorded as positive or negative using control wells for color comparison. If the color change was darker than the negative control, the sample was considered positive. Milk zinc. For each of the 107 study days December 15, 2015 to March 31, 2016 of zinc supplementation, approximately 1.5 mL of treated milk from two bottles of each treatment group were randomly collected into 2.0 mL microcentrifuge tubes with polypropylene snap caps Fisher Scientific, Pittsburgh, PA , and stored at −20˚C until analysis.", "Milk zinc. For each of the 107 study days December 15, 2015 to March 31, 2016 of zinc supplementation, approximately 1.5 mL of treated milk from two bottles of each treatment group were randomly collected into 2.0 mL microcentrifuge tubes with polypropylene snap caps Fisher Scientific, Pittsburgh, PA , and stored at −20˚C until analysis. At the time of analysis, milk samples were thawed at 4˚C, vortexed, pooled by week and treatment group, and analyzed for zinc concentration ppm by ICP-MS inductively coupled plasma mass spectrometry at the CAHFS Laboratory. Quality control samples, including method blanks, laboratory control spikes, National Institute of Standards and Technology NIST reference materials NIST 1640 , and a spiked milk sample, were run with each set of study samples. Data analysis was performed using R Statistic Software version 3.3.1 and Stata IC 14.2 College Station, TX . Statistical differences were determined at the 5% level of significance using per protocol analysis.", "Data analysis was performed using R Statistic Software version 3.3.1 and Stata IC 14.2 College Station, TX . Statistical differences were determined at the 5% level of significance using per protocol analysis. An ANOVA was used to compare calves in each treatment group at enrollment with respect to birth weight kg , serum total protein g/dL , attitude score, and fecal score. Oral zinc dose at the start and end of treatment was calculated as the zinc supplementation dose 80 mg divided by calf body weight kg at birth and on the last day of treatment, respectively. An ANOVA was used to compare oral zinc dose mg/kg at treatment start and end as well as mean body weight kg at birth, end of treatment, and hutch exit between treatment groups and between bulls and heifers. A Chi-Square test of Independence was used to compare the proportions of calves by sex as well as mortality between treatment groups.", "An ANOVA was used to compare oral zinc dose mg/kg at treatment start and end as well as mean body weight kg at birth, end of treatment, and hutch exit between treatment groups and between bulls and heifers. A Chi-Square test of Independence was used to compare the proportions of calves by sex as well as mortality between treatment groups. For all analyses, Tukey's Honest Significant Difference method was used to generate pairwise comparisons to further characterize significant differences identified by ANOVA. Residual diagnostics, including Residuals vs. Fitted, Scale-Location, Normal Q-Q, and Cook's distances plots, were used to validate all ANOVA model assumptions. The non-parametric Kruskal-Wallis Rank Sum test was used when assumptions were violated. For the randomly-sampled calves, Fisher exact tests were used to compare fecal pathogen prevalence on the first day of diarrhea and at diarrhea resolution between treatment groups.", "The non-parametric Kruskal-Wallis Rank Sum test was used when assumptions were violated. For the randomly-sampled calves, Fisher exact tests were used to compare fecal pathogen prevalence on the first day of diarrhea and at diarrhea resolution between treatment groups. Pairwise comparisons with Bonferroni adjustment were used to identify specific differences in pathogen prevalence. An ANOVA was used to compare serum zinc concentration before and after treatment between treatment groups and between bulls and heifers. A Kruskal-Wallis Rank Sum test was used to compare rank sums of zinc concentrations in pooled milk samples from different treatment groups. Post-hoc Nemenyi-tests for pairwise multiple comparisons of ranked data were used to identify specific differences in zinc concentrations between groups.", "A Kruskal-Wallis Rank Sum test was used to compare rank sums of zinc concentrations in pooled milk samples from different treatment groups. Post-hoc Nemenyi-tests for pairwise multiple comparisons of ranked data were used to identify specific differences in zinc concentrations between groups. For all regression models in this study, univariate regression was first used to evaluate associations between individual predictor variables and outcomes. All variables with statistical and/or biological significance were initially included in multivariate regression models. The final models were built using a manual backwards elimination procedure, with a significance level of P > 0.05 as the removal criterion. Confounding was assessed using the method of change of estimates, where a 10% or greater change in the estimate of the treatment group regression coefficient between the models with and without the confounder variable was used as evidence of confounding .", "The final models were built using a manual backwards elimination procedure, with a significance level of P > 0.05 as the removal criterion. Confounding was assessed using the method of change of estimates, where a 10% or greater change in the estimate of the treatment group regression coefficient between the models with and without the confounder variable was used as evidence of confounding . Variables identified as confounders were included in the final model. All possible interactions between treatment group and predictor variables were explored and retained in the final model if statistically significant. Microbiological cure. For the randomly-sampled calves, logistic regression was used to evaluate associations between microbiological cure and treatment group.", "Microbiological cure. For the randomly-sampled calves, logistic regression was used to evaluate associations between microbiological cure and treatment group. Other predictor variables of interest included sex, serum total protein, and age on the first day of diarrhea. Microbiological cure was defined as a negative fecal ELISA test at resolution of clinical diarrhea for calves with a positive ELISA test on the first day of diarrhea for at least one of the four fecal pathogens E. coli K99, bovine rotavirus and coronavirus, and C. parvum . Models were generated for each fecal pathogen individually and an overall model, which evaluated microbiological cure at clinical diarrhea resolution for calves that tested positive for any single pathogen on the first day of diarrhea. Serum total protein and calf age at first diarrhea were included in all final models to control for potential confounding by passive transfer status and age.", "Models were generated for each fecal pathogen individually and an overall model, which evaluated microbiological cure at clinical diarrhea resolution for calves that tested positive for any single pathogen on the first day of diarrhea. Serum total protein and calf age at first diarrhea were included in all final models to control for potential confounding by passive transfer status and age. Mean daily weight change. Linear regression was used to evaluate associations between ADG kg and treatment group during the treatment and pre-weaning periods separately. Other predictor variables of interest included sex, birth weight kg , serum total protein, number of days having diarrhea, age, and volume L of milk, Calva, and NuLife electrolytes fed at either end of treatment or hutch exit. For each calf, ADG during the treatment and pre-weaning period was calculated as the difference between birth and end treatment or hutch exit weight, respectively, divided by the number of days between these time points.", "Other predictor variables of interest included sex, birth weight kg , serum total protein, number of days having diarrhea, age, and volume L of milk, Calva, and NuLife electrolytes fed at either end of treatment or hutch exit. For each calf, ADG during the treatment and pre-weaning period was calculated as the difference between birth and end treatment or hutch exit weight, respectively, divided by the number of days between these time points. To explore the possibility of an interaction between treatment group, sex, and birth weight, the final linear regression model for ADG during the pre-weaning period was stratified by sex. Age at the end of treatment or hutch exit and number of days with diarrhea were dropped from all final models in favor of improved Akaike information criterion AIC . Onset of diarrhea and clinical cure. For all survival analyses, diarrhea was defined as a fecal score greater than 1 while diarrhea cure was defined as the second consecutive day of normal feces fecal score of 1 following the first diarrhea episode.", "Onset of diarrhea and clinical cure. For all survival analyses, diarrhea was defined as a fecal score greater than 1 while diarrhea cure was defined as the second consecutive day of normal feces fecal score of 1 following the first diarrhea episode. Subsequent episodes of diarrhea were not included in the analysis. Calves that died or did not experience diarrhea or cure from diarrhea were censored. If fresh feces were not observed on daily calf hutch assessment, a fecal score was not recorded for that day and not included in the analyses. Kaplan-Meier analysis was used to determine median days to first diarrhea event and, for those calves that developed diarrhea during the assessment period, median days to clinical diarrhea cure.", "If fresh feces were not observed on daily calf hutch assessment, a fecal score was not recorded for that day and not included in the analyses. Kaplan-Meier analysis was used to determine median days to first diarrhea event and, for those calves that developed diarrhea during the assessment period, median days to clinical diarrhea cure. A Log Rank test of equality was used to compare survivor functions between treatments. Cox Proportional Hazards regression analysis was used to estimate and compare the hazard of diarrhea and diarrhea cure between treatment groups. Sex, age, serum total protein at enrollment, birth weight kg , antimicrobial therapy, and application of fresh straw to the hutches were evaluated as predictor variables and potential confounders. When modeling the hazard of diarrhea cure, a binary variable termed therapeutic supplementation indicating whether calves were treated with either ZM, ZS or placebo for all or at least the first 5 days of diarrhea was evaluated as an additional covariate.", "Sex, age, serum total protein at enrollment, birth weight kg , antimicrobial therapy, and application of fresh straw to the hutches were evaluated as predictor variables and potential confounders. When modeling the hazard of diarrhea cure, a binary variable termed therapeutic supplementation indicating whether calves were treated with either ZM, ZS or placebo for all or at least the first 5 days of diarrhea was evaluated as an additional covariate. A five-day period was selected by the authors based on clinical experience, as five days represents a reasonable duration over which most therapeutic treatments for calf diarrhea should be applied and be expected to alleviate disease. The proportional hazards assumption that the hazard of diarrhea is independent of time was assessed using analysis of Schoenfeld residuals and testing whether the log hazard-ratio function is constant over time. Any variable found to violate the proportional hazards assumption was included in the final regression model as a time varying covariate. A total of 1,513 calves were enrolled in the trial.", "Any variable found to violate the proportional hazards assumption was included in the final regression model as a time varying covariate. A total of 1,513 calves were enrolled in the trial. However, due to failure to immediately recognize exclusion criteria, 23 calves were excluded shortly after enrollment. In addition, 8 calves were excluded due to treatment errors. Therefore, a total of 1,482 calves placebo = 500, ZM = 491, ZS = 491 were included in the final analyses. A total of 242 calves 16.3% had minimal fecal output at the time of enrollment while 125 calves 8.4% had abnormal fecal scores of 2 or 3 that were described as meconium.", "Therefore, a total of 1,482 calves placebo = 500, ZM = 491, ZS = 491 were included in the final analyses. A total of 242 calves 16.3% had minimal fecal output at the time of enrollment while 125 calves 8.4% had abnormal fecal scores of 2 or 3 that were described as meconium. All enrolled calves appeared healthy on visual assessment and hence were assumed to have a normal fecal score at the time of enrollment. The three treatment groups at enrollment did not differ significantly in mean birth weight kg P = 0.244 , mean serum total protein g/dL P = 0.541 , mean attitude score P = 0.845 , mean fecal score P = 0.522 , as shown in Table 1 , or distribution of calf sex P = 0.472 . Of the 1,482 study calves, 21 1.4% died during the trial: 5 calves in the placebo group 1.0% , 11 calves in the ZM group 2.2% , and 5 calves in the ZS group 1.0% . Of these 21 calves that died, 14 66.7% were bulls and 7 33.3% were heifers.", "Of the 1,482 study calves, 21 1.4% died during the trial: 5 calves in the placebo group 1.0% , 11 calves in the ZM group 2.2% , and 5 calves in the ZS group 1.0% . Of these 21 calves that died, 14 66.7% were bulls and 7 33.3% were heifers. Eighteen of the 21 calves 85.7% were found dead, rather than euthanized, due to acute and spontaneous death without previous obvious clinical signs of disease. The remaining 3 calves were euthanized prior to death due to severe and/or prolonged morbidity. Characteristics and causes of death based on field necropsy of these calves can be found in S4 Table. There was no significant difference in the proportion of calves that died between treatment groups P = 0.168 , though mortality was significantly higher in bulls compared to heifer calves P = 0.049 .", "Characteristics and causes of death based on field necropsy of these calves can be found in S4 Table. There was no significant difference in the proportion of calves that died between treatment groups P = 0.168 , though mortality was significantly higher in bulls compared to heifer calves P = 0.049 . Birth weight data were available for all 1,482 calves. Due to calf mortality between enrollment and completion of treatment n = 4 , end treatment weight data were available for 1,478 calves. Similarly, due to calf mortality n = 21 or missing data for body weight at hutch exit from the dairy's records n = 40 , hutch exit weight data were available for 1,421 calves. A summary of body weight data stratified by treatment group and sex is presented in Table 2 .", "Similarly, due to calf mortality n = 21 or missing data for body weight at hutch exit from the dairy's records n = 40 , hutch exit weight data were available for 1,421 calves. A summary of body weight data stratified by treatment group and sex is presented in Table 2 . Within each treatment group, bull calves showed consistently higher birth weight P < 0.001 , end treatment weight P < 0.001 , and exit hutch weight P < 0.001 compared to heifer calves. However, at birth, end of treatment, or hutch exit there were no differences in body weight between treatment groups for bulls P > 0.1 or heifers P > 0.1 . The mean attitude scores during the study period were 1.2 across the three treatment groups P = 0.208 . Of 1,482 calves included in the final analysis, A total of 629 treated milk samples were obtained throughout the 107-day study period and pooled by treatment group and week, yielding a total of 16 pooled samples per treatment group.", "The mean attitude scores during the study period were 1.2 across the three treatment groups P = 0.208 . Of 1,482 calves included in the final analysis, A total of 629 treated milk samples were obtained throughout the 107-day study period and pooled by treatment group and week, yielding a total of 16 pooled samples per treatment group. Zinc concentrations ppm were significantly higher in pooled milk samples treated with ZM P < 0.001 and ZS P < 0.001 compared to placebo-treated samples, and there were no significant differences between ZM and ZS-treated samples P = 1.000 , as shown in S5 Table. Within zinc treatment groups, oral zinc dose at the start and end of treatment is summarized by sex in S6 Table. For both ZM-and ZS-treated calves, oral zinc dose at the start P < 0.001 and end P < 0.001 of treatment was significantly higher in heifer versus bull calves. Serum zinc concentrations before and after treatment were obtained from 36 calves n = 12 for each treatment group and are summarized in S7 Table.", "For both ZM-and ZS-treated calves, oral zinc dose at the start P < 0.001 and end P < 0.001 of treatment was significantly higher in heifer versus bull calves. Serum zinc concentrations before and after treatment were obtained from 36 calves n = 12 for each treatment group and are summarized in S7 Table. Overall, there were no significant differences in mean pre-treatment serum zinc concentrations between treatment groups P = 0.233 . Mean post-treatment serum zinc concentrations were significantly higher in calves treated with ZM P < 0.001 and ZS P = 0.002 compared to placebo-treated calves, and there were no significant differences among calves treated with ZM and ZS P = 0.406 . Stratification of serum zinc data by treatment group and sex demonstrated that for ZM-treated calves, heifers had a numerically higher post-treatment serum zinc concentration compared to bulls, though the difference was not statistically significant P = 0.199 . In contrast, in ZStreated calves, heifers had a numerically lower post-treatment serum zinc concentration compared to bulls, though the difference was also not statistically significant P = 0.538 .", "Stratification of serum zinc data by treatment group and sex demonstrated that for ZM-treated calves, heifers had a numerically higher post-treatment serum zinc concentration compared to bulls, though the difference was not statistically significant P = 0.199 . In contrast, in ZStreated calves, heifers had a numerically lower post-treatment serum zinc concentration compared to bulls, though the difference was also not statistically significant P = 0.538 . Fecal analysis data were analyzed for 92 of the 127 randomly-selected calves. The remaining 35 calves were not included in the analysis due to not acquiring diarrhea during the assessment period n = 10 , death prior to final sampling n = 1 , exclusion due to improper treatment regimen n = 1 , or incorrect sampling day n = 23 .The 92 calves had a mean age at onset of diarrhea of 13.3, 11.0 and 11.3 days for ZM, ZS and placebo-treated groups, respectively; and a mean age at resolution of diarrhea of 18.8, 16.1 and 15.7 days for ZM, ZS and placebo-treated groups, respectively. There were no significant differences in the prevalence of E. coli K99 P = 0.694 , rotavirus P = 0.331 , coronavirus P = 0.819 , or C. parvum P = 0.719 fecal shedding on the first day of diarrhea between treatment groups S8 Table . There were no significant differences in the prevalence of E. coli K99 P = 0.256 , rotavirus P = 0.344 , or coronavirus P = 1.000 fecal shedding at resolution of diarrhea between treatment groups, though there was a difference in C. parvum P = 0.006 fecal shedding between treatment groups S9 Table .", "There were no significant differences in the prevalence of E. coli K99 P = 0.694 , rotavirus P = 0.331 , coronavirus P = 0.819 , or C. parvum P = 0.719 fecal shedding on the first day of diarrhea between treatment groups S8 Table . There were no significant differences in the prevalence of E. coli K99 P = 0.256 , rotavirus P = 0.344 , or coronavirus P = 1.000 fecal shedding at resolution of diarrhea between treatment groups, though there was a difference in C. parvum P = 0.006 fecal shedding between treatment groups S9 Table . The prevalence of C. parvum fecal shedding at resolution of diarrhea was significantly higher in calves treated with ZM P = 0.009 and ZS P = 0.023 compared to placebo-treated calves, and there were no significant differences among calves treated with ZM and ZS P = 1.000 . Results of logistic regression models for overall and pathogen-specific microbiological cure are presented in Tables 3-5 . For pathogen-specific cure, all calves that tested positive on the first day of diarrhea for coronavirus n = 4 tested negative at clinical diarrhea resolution. For calves that tested positive on the first day of diarrhea for E. coli K99 n = 9 , all placebo-treated calves n = 2 tested negative at clinical diarrhea resolution while half n = 2 of all ZS-treated calves tested either positive or negative at clinical diarrhea resolution, resulting in omission of ZS treatment variable from the model due to collinearity.", "For pathogen-specific cure, all calves that tested positive on the first day of diarrhea for coronavirus n = 4 tested negative at clinical diarrhea resolution. For calves that tested positive on the first day of diarrhea for E. coli K99 n = 9 , all placebo-treated calves n = 2 tested negative at clinical diarrhea resolution while half n = 2 of all ZS-treated calves tested either positive or negative at clinical diarrhea resolution, resulting in omission of ZS treatment variable from the model due to collinearity. Hence, logistic regression analyses for microbiological cure in calves that tested positive for coronavirus and E. coli K99 were not possible. For 59 calves that tested positive for rotavirus on the first day of diarrhea Table 3 , calves treated with ZM had a 50% increased odds of testing negative at diarrhea resolution compared to placebo-treated calves, though this difference was not significant P = 0.549 . Likewise, calves treated with ZS had 100% increased odds 2 times the odds of testing negative for rotavirus at diarrhea resolution compared to placebo-treated calves, though this difference was also not significant P = 0.314 . However, this model demonstrated a significant main effect of serum total protein, such that for every 1 unit g/dL increase in serum total protein at enrollment, the odds of microbiological cure of rotavirus decreased by 79% P = 0.026 .", "Likewise, calves treated with ZS had 100% increased odds 2 times the odds of testing negative for rotavirus at diarrhea resolution compared to placebo-treated calves, though this difference was also not significant P = 0.314 . However, this model demonstrated a significant main effect of serum total protein, such that for every 1 unit g/dL increase in serum total protein at enrollment, the odds of microbiological cure of rotavirus decreased by 79% P = 0.026 . For 40 calves that tested positive for Cryptosporidium parvum on the first day of diarrhea Table 4 , calves treated with ZM had an 87% reduced odds of testing negative at diarrhea resolution Effect of zinc on diarrhea in calves compared to placebo-treated calves, though this difference was not significant P = 0.119 . Likewise, calves treated with ZS had a 74% reduced odds of testing negative for Cryptosporidium parvum at diarrhea resolution compared to placebo-treated calves, though this difference was also not significant P = 0.183 . For 55 calves that tested positive for any one of the four fecal pathogens E. coli K99, rotavirus, coronavirus, Cryptosporidium parvum on the first day of diarrhea Table 5 , calves treated with ZM had a 25% increased odds of testing negative at diarrhea resolution compared to placebo-treated calves, though this difference was not significant P = 0.769 . Likewise, calves treated with ZS had a 52% increased odds of testing negative for Cryptosporidium parvum at diarrhea resolution compared to placebo-treated calves, though this difference was also not significant P = 0.633 .", "For 55 calves that tested positive for any one of the four fecal pathogens E. coli K99, rotavirus, coronavirus, Cryptosporidium parvum on the first day of diarrhea Table 5 , calves treated with ZM had a 25% increased odds of testing negative at diarrhea resolution compared to placebo-treated calves, though this difference was not significant P = 0.769 . Likewise, calves treated with ZS had a 52% increased odds of testing negative for Cryptosporidium parvum at diarrhea resolution compared to placebo-treated calves, though this difference was also not significant P = 0.633 . However, this model demonstrated that heifer calves had 71% lower odds of microbiological cure of any single fecal pathogen compared to bull calves P = 0.076 . A total of 1,482 calves were included in the linear regression model results for ADG during the treatment period, which are presented in Table 6 . There was no significant difference in ADG for ZM-or ZS-treated calves compared to placebo-treated calves, though there were significant main effects of sex, birth weight, and milk volume. Specifically, heifer calves gained 70 g bodyweight per day less compared to bull calves P < 0.001 .", "There was no significant difference in ADG for ZM-or ZS-treated calves compared to placebo-treated calves, though there were significant main effects of sex, birth weight, and milk volume. Specifically, heifer calves gained 70 g bodyweight per day less compared to bull calves P < 0.001 . For every 1 kg increase in birth weight, calves gained 16 g per day less than their herd mates P < 0.001 . For every 1 L increase in milk volume per day during the treatment period, calves gained an additional 13 g per day P < 0.001 . Table 7 summarizes the linear regression analysis of ADG during the pre-weaning period for 1,421 calves which showed a significant difference in ADG for ZM-treated calves compared to placebo-treated calves and in bull versus heifer calves. Milk volume had a significant effect on ADG, such that for every 1 L increase in milk volume per day during the treatment period, calves gained an additional 2 g bodyweight per day P = 0.001 .", "Table 7 summarizes the linear regression analysis of ADG during the pre-weaning period for 1,421 calves which showed a significant difference in ADG for ZM-treated calves compared to placebo-treated calves and in bull versus heifer calves. Milk volume had a significant effect on ADG, such that for every 1 L increase in milk volume per day during the treatment period, calves gained an additional 2 g bodyweight per day P = 0.001 . Results of the final model showed a significant main effect for ZM-treated bull calves and a significant interaction term for ZM treatment by sex. After controlling for milk volume received during the treatment Table 6 calves n = 1,482 Effect of zinc on diarrhea in calves period, ZM-treated bulls gained 22 g body weight per day on average more than placebotreated bull calves P = 0.042 and ZM-treated heifers gained 12 g less body weight per day on average compared to placebo-treated heifers P = 0.019 . When considering the model coefficients for treatment group, sex, and their interaction, bull calves treated with ZM gained 454 g per day 0.432 + 0.022 while female calves treated with ZM gained 0.404 g per day 0.432 + 0.022-0.016-0.034 , hence 50 g per day more gain in male calves compared to heifers treated with ZM P = 0.019 . For ZS-treated calves, there was a numerical decrease in weight gain of 5 g per day in bulls and 11 g per day in heifers compared to placebo-treated calves, though the differences were not significant P = 0.673 bulls; P = 0.681 heifers .", "When considering the model coefficients for treatment group, sex, and their interaction, bull calves treated with ZM gained 454 g per day 0.432 + 0.022 while female calves treated with ZM gained 0.404 g per day 0.432 + 0.022-0.016-0.034 , hence 50 g per day more gain in male calves compared to heifers treated with ZM P = 0.019 . For ZS-treated calves, there was a numerical decrease in weight gain of 5 g per day in bulls and 11 g per day in heifers compared to placebo-treated calves, though the differences were not significant P = 0.673 bulls; P = 0.681 heifers . Linear regression models of ADG during the pre-weaning period were stratified by sex in order to avoid interpreting a three-way interaction between treatment group, sex, and birth weight. In the heifer model S10 Table , the interaction between ZM treatment and birth weight was significant which implied that birth weight modified the effect of ZM treatment on ADG. At a 29 kg birth weight two standard deviations below the mean , ZM-treated heifers gained 49 g body weight per day on average less than placebo-treated heifers P = 0.037 . However, at a 49 kg birth weight two standard deviations above the mean , ZM-treated heifers gained 30 g body weight per day on average more than placebo-treated heifers P = 0.037 .", "At a 29 kg birth weight two standard deviations below the mean , ZM-treated heifers gained 49 g body weight per day on average less than placebo-treated heifers P = 0.037 . However, at a 49 kg birth weight two standard deviations above the mean , ZM-treated heifers gained 30 g body weight per day on average more than placebo-treated heifers P = 0.037 . In the bull calves model S11 Table there was no significant interaction between treatment group and birth weight. A total of 1,482 calves were included in the Kaplan-Meier survival analysis of time to first diarrhea event Fig 1 . There were no significant differences in median age at onset of diarrhea, specifically, 8, 8 and 7 days for the ZM, ZS and placebo-treated calves, respectively P = 0.402 . Cox proportional hazard regression model for diarrhea hazard are presented in Table 8 .", "There were no significant differences in median age at onset of diarrhea, specifically, 8, 8 and 7 days for the ZM, ZS and placebo-treated calves, respectively P = 0.402 . Cox proportional hazard regression model for diarrhea hazard are presented in Table 8 . After controlling for age, calves treated with ZM had a 14.7% reduced hazard of diarrhea compared to placebo-treated calves P = 0.015 . Calves treated with ZS had 13.9% reduced hazard of diarrhea compared to placebo-treated calves P = 0.022 . calves n = 1,421 A total of 1,394 calves were included in the Kaplan-Meier survival analysis of time to clinical diarrhea cure Fig 2 , as 88 calves failed to acquire diarrhea during the assessment period. There were no significant differences in the median days to diarrhea cure which was 7 days across all 3 treatment groups P = 0.264 .", "calves n = 1,421 A total of 1,394 calves were included in the Kaplan-Meier survival analysis of time to clinical diarrhea cure Fig 2 , as 88 calves failed to acquire diarrhea during the assessment period. There were no significant differences in the median days to diarrhea cure which was 7 days across all 3 treatment groups P = 0.264 . Cox proportional hazard regression model for diarrhea cure hazard are presented in Table 9 . Results of the final model showed a significant interaction term between treatment and therapeutic supplementation as well as the need for age as a time varying covariate. When considering calves that did not receive supplementation, respective to each of the 3 groups, for at least the first five days of diarrhea there was no significant difference between either ZM-and ZS-treated calves compared to placebo-treated calves P = 0.223 ZM, P = 0.134 ZS . However, when considering calves that were supplemented for at least the first five days of diarrhea, ZM-treated calves experienced a 21.4% higher hazard of cure from diarrhea compared to placebo-treated calves P = 0.027 .", "When considering calves that did not receive supplementation, respective to each of the 3 groups, for at least the first five days of diarrhea there was no significant difference between either ZM-and ZS-treated calves compared to placebo-treated calves P = 0.223 ZM, P = 0.134 ZS . However, when considering calves that were supplemented for at least the first five days of diarrhea, ZM-treated calves experienced a 21.4% higher hazard of cure from diarrhea compared to placebo-treated calves P = 0.027 . Likewise, ZS-treated calves experienced a 13.0% higher hazard of cure from diarrhea compared to placebo-treated calves P = 0.040 . The current trial demonstrated evidence for the beneficial effect of ZM on ADG and neonatal diarrhea as well as an effect of ZS on diarrhea in dairy calves during the pre-weaning period. It is important to consider these results in the context of the entire pre-weaning and hutch period. On average, after 90 days from birth to hutch exit, placebo-treated bull calves gained 38.88 kg body weight while ZM-treated bull calves gained an additional 1.98 kg 40.86 kg .", "It is important to consider these results in the context of the entire pre-weaning and hutch period. On average, after 90 days from birth to hutch exit, placebo-treated bull calves gained 38.88 kg body weight while ZM-treated bull calves gained an additional 1.98 kg 40.86 kg . In contrast, the effect of zinc on weight gain in treated heifers depended on birth weight. Low birth weight heifers treated with ZM gained on average less than a placebo-treated heifer of the same birth weight. In contrast, high birth weight heifers treated with ZM gained more than placebo-treated heifers of the same birth weight. The switch in direction of the association between ZM treatment and ADG in heifer calves depending on birth weight suggests a doseresponse effect rather than a true sex-specific effect of ZM on ADG.", "In contrast, high birth weight heifers treated with ZM gained more than placebo-treated heifers of the same birth weight. The switch in direction of the association between ZM treatment and ADG in heifer calves depending on birth weight suggests a doseresponse effect rather than a true sex-specific effect of ZM on ADG. Hence, low birth weight calves including heifers may require a lower dose of ZM to mitigate any negative effect of what is otherwise a suitable dose for higher birth weight calves. These findings are in agreement with a previous randomized clinical trial testing the effect of daily oral zinc in diarrheic neonatal Holstein calves which, showed that ZM-treated calves had a numerically, though not significantly increased ADG compared to calves treated with zinc oxide or placebo due to small sample size . In general, our trial findings are in agreement with the large body of human literature supporting the use of oral zinc for the prevention and treatment of diarrhea and impaired growth in children . Zinc supplementation is widely accepted by global health organizations as a vital component of therapy for childhood diarrhea , however, recent reviews of the literature demonstrated heterogeneity in study results on the basis of age, baseline zinc status, geographic location, and supplementation regimen .", "In general, our trial findings are in agreement with the large body of human literature supporting the use of oral zinc for the prevention and treatment of diarrhea and impaired growth in children . Zinc supplementation is widely accepted by global health organizations as a vital component of therapy for childhood diarrhea , however, recent reviews of the literature demonstrated heterogeneity in study results on the basis of age, baseline zinc status, geographic location, and supplementation regimen . Similar to our findings, a sex-specific response to zinc supplementation has been demonstrated in several human studies. Zinc gluconate administered for diarrhea prevention reduced the incidence of dysentery in treated boys but not girls ; when given therapeutically, it reduced diarrhea duration and frequency more dramatically in boys compared to girls . Similarly, zinc sulfate was shown to improve Effect of zinc on diarrhea in calves diarrhea outcomes in boys but improved growth rates in girls . Broadly, these differences between male and female responses to zinc supplementation are not understood, though theories regarding differences in immune function and response , diarrhea etiology , and nutrient requirements have been proposed.", "Similarly, zinc sulfate was shown to improve Effect of zinc on diarrhea in calves diarrhea outcomes in boys but improved growth rates in girls . Broadly, these differences between male and female responses to zinc supplementation are not understood, though theories regarding differences in immune function and response , diarrhea etiology , and nutrient requirements have been proposed. In the current study, ZM-treated bulls demonstrated increased ADG compared to placebo-treated bulls while ZM-treated heifers demonstrated decreased ADG compared to placebo-treated heifers. However, due to a significant interaction between ZM treatment and birth weight, this reduction in ADG in ZMtreated heifers was overcome with increasing birth weight, such that ZM-treated heifers with birth weights above 42 kg experienced increased ADG during the pre-weaning period, compared to placebo-treated heifers with birth weights above 42 kg. Differences in the growth response to ZM supplementation between bull and heifer calves may have been related to its effect on feed intake. Previous research on the effects of feeding various doses of oral zinc oxide to pre-ruminant dairy calves demonstrated that high levels of oral zinc supplementation resulted in reduced feed intake .", "Differences in the growth response to ZM supplementation between bull and heifer calves may have been related to its effect on feed intake. Previous research on the effects of feeding various doses of oral zinc oxide to pre-ruminant dairy calves demonstrated that high levels of oral zinc supplementation resulted in reduced feed intake . In the current trial, oral ZM dose was estimated to be significantly higher in heifers compared to bulls due to the significantly lower birth weight of heifers. Additionally, serum zinc concentrations in ZM-treated heifers were numerically higher than that of bulls, though this difference was not significant, likely due to the small sample size. Perhaps the higher zinc dose in heifers was associated with reduced feed intake, leading to reduced growth, and that this effect was more pronounced for ZM compared to ZS. The fact that ZM-treated heifers with birth weights approaching those of average bull calves and, therefore, a similar zinc dose to that in bulls experienced an increase in ADG over placebo-treated heifers similar to that of bull calves partially supports this theory.", "Perhaps the higher zinc dose in heifers was associated with reduced feed intake, leading to reduced growth, and that this effect was more pronounced for ZM compared to ZS. The fact that ZM-treated heifers with birth weights approaching those of average bull calves and, therefore, a similar zinc dose to that in bulls experienced an increase in ADG over placebo-treated heifers similar to that of bull calves partially supports this theory. Although management practices on the study dairy were designed to be identical for both bulls and heifers, it is possible that subtle, unrecognized differences in nutritional and health management may also have contributed to sex-specific differences in weight gain. Nevertheless, future trials are warranted to investigate the potential differences in the dose-response to zinc supplementation between bulls and heifers. We hypothesized that ADG would be increased in zinc-supplemented calves compared to placebo-supplemented calves due to the potential preventive and therapeutic effects of zinc supplementation on neonatal diarrhea. In other words, calf diarrhea is mitigated by zinc supplementation and, therefore, on the causal pathway between zinc and ADG.", "We hypothesized that ADG would be increased in zinc-supplemented calves compared to placebo-supplemented calves due to the potential preventive and therapeutic effects of zinc supplementation on neonatal diarrhea. In other words, calf diarrhea is mitigated by zinc supplementation and, therefore, on the causal pathway between zinc and ADG. However, considering the similarly-reduced hazard of diarrhea and increased hazard of cure from diarrhea in both ZM and ZS treatment groups but a lack of effect of ZS on ADG, it is likely that the effect of ZM on ADG is not solely mediated through its effects on diarrhea. Differences in effectiveness between organic and inorganic formulations also may exist. In fact, the underlying mechanism of action of oral zinc remains unknown . Several theories of the mechanisms of action of zinc in the prevention and treatment of childhood diarrhea exist, including a mucosal-protective role, a diarrhea-induced zinc deficiency, an essential element in cell-mediated immunity, and a modifier of intra-luminal electrolyte secretion and absorption 6, .", "In fact, the underlying mechanism of action of oral zinc remains unknown . Several theories of the mechanisms of action of zinc in the prevention and treatment of childhood diarrhea exist, including a mucosal-protective role, a diarrhea-induced zinc deficiency, an essential element in cell-mediated immunity, and a modifier of intra-luminal electrolyte secretion and absorption 6, . The clinical and practical implications of effects of ZM supplementation on ADG and diarrhea must be considered. Pre-weaned calf diarrhea remains an ongoing issue for the dairy industry. The deleterious effects on calf health and performance and the resulting economic burden create a strong incentive to treat and prevent diarrhea in pre-weaned calves. On large dairy operations like those in California's Central Valley, small changes in disease incidence and duration as well as animal growth and performance can have profound economic consequences.", "The deleterious effects on calf health and performance and the resulting economic burden create a strong incentive to treat and prevent diarrhea in pre-weaned calves. On large dairy operations like those in California's Central Valley, small changes in disease incidence and duration as well as animal growth and performance can have profound economic consequences. As a non-antimicrobial product, zinc may become increasingly attractive as antimicrobials in livestock feed are under increased scrutiny and regulation due to concerns about antimicrobial resistance . Prevalence of C. parvum fecal shedding in a random sample of 92 study calves at onset and resolution of diarrhea was significantly higher in calves treated with zinc compared to Placebo-treated calves. In contrast, a previous study where calves that tested positive for C. parvum at the start of diarrhea and were treated with ZM had 16 times higher odds of being fecal ELISA negative at exit compared to the Placebo group P = 0.08; power = 72.3% . The difference in findings may be due to the differences in the timing of diarrhea across treatment groups.", "In contrast, a previous study where calves that tested positive for C. parvum at the start of diarrhea and were treated with ZM had 16 times higher odds of being fecal ELISA negative at exit compared to the Placebo group P = 0.08; power = 72.3% . The difference in findings may be due to the differences in the timing of diarrhea across treatment groups. For the current study's random sample of calves that acquired, survived, and were sampled on the correct days, the mean age of calves on both onset and resolution of diarrhea was higher for ZM and ZS calves compared to placebo-treated calves. Although C. parvum oocyst shedding in infected calves can occur as early as 3 days of age, peak shedding occurs at about 14 days of age . It is possible that the increase in prevalence of C. parvum shedding in ZM and ZS treated groups was due to the increased age of zinc-treated calves compared to placebo-treated calves at resolution of diarrhea. The latter explanation is also supported by our findings that the odds of microbiological cure from C. parvum significantly decreased in older calves, with no significant differences in the odds of cure between treatment groups.", "It is possible that the increase in prevalence of C. parvum shedding in ZM and ZS treated groups was due to the increased age of zinc-treated calves compared to placebo-treated calves at resolution of diarrhea. The latter explanation is also supported by our findings that the odds of microbiological cure from C. parvum significantly decreased in older calves, with no significant differences in the odds of cure between treatment groups. In addition, the current testing did not estimate the concentration of C. parvum shedding which may still differ between treatment groups. Despite the large sample size, the current trial was limited to a single California dairy, which may represent other large dairies but does not reflect all the dairy management systems in California or elsewhere. Additionally, our results show that calves respond to zinc supplementation for diarrhea prevention differently depending on chemical formulation and calf sex. The latter could be due to differences in body weight between bulls and heifers and may point towards the need for sex-specific dosing.", "Additionally, our results show that calves respond to zinc supplementation for diarrhea prevention differently depending on chemical formulation and calf sex. The latter could be due to differences in body weight between bulls and heifers and may point towards the need for sex-specific dosing. Furthermore, the current research did not evaluate the potential economic utility of zinc supplementation. Future studies on more accurate dosing of zinc by calf sex, the practical feasibility of weight-based dosing, and the expected cost-effectiveness of zinc administration as part of the management of pre-weaned dairy calves are warranted. Finally, our clinical trial was performed on a single, large, predominately Holstein, California dairy over a six-month period, which precluded our ability to evaluate differences due to season or breed. Hence, future studies to assess any modifying effect of breed and seasonal differences on the effect of zinc on calf health and weight gain are also needed.", "Finally, our clinical trial was performed on a single, large, predominately Holstein, California dairy over a six-month period, which precluded our ability to evaluate differences due to season or breed. Hence, future studies to assess any modifying effect of breed and seasonal differences on the effect of zinc on calf health and weight gain are also needed. The current double blind, block-randomized placebo controlled clinical trial tested the effect of a prophylactic daily oral zinc supplementation in neonatal Holstein calves. Bull calves treated with ZM had a significantly increased ADG 22 g per day during the pre-weaning period compared to placebo-treated bulls. In comparison, ZM-treated heifers had significantly lower average daily gain 9 g per day compared to placebo-treated heifers, although higher ZM doses in low birthweight heifers may explain the lower ADG. Calves treated with either ZM or ZS had significantly lower risks of diarrhea and significantly higher risk of cure from diarrhea over the first 30 days of life compared to placebo-treated calves and hence the current trial demonstrated that zinc supplementation delayed diarrhea and expedited diarrhea recovery in pre-weaned calves.", "In comparison, ZM-treated heifers had significantly lower average daily gain 9 g per day compared to placebo-treated heifers, although higher ZM doses in low birthweight heifers may explain the lower ADG. Calves treated with either ZM or ZS had significantly lower risks of diarrhea and significantly higher risk of cure from diarrhea over the first 30 days of life compared to placebo-treated calves and hence the current trial demonstrated that zinc supplementation delayed diarrhea and expedited diarrhea recovery in pre-weaned calves. Additionally, zinc improved weight gain differentially in bulls compared to heifers, indicating the need for further research to investigate zinc dosing in calves. Supporting information S1 DOCX S1 Dataset. Raw data collected from trial, organized as separate excel sheets for enrollment, daily assessment, serum total protein, birth weight, exit treatment weight, exit trial weight, serum zinc testing, fecal samples, fecal testing, milk testing, and dead calves. XLSX" ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]

[ "The continued emergence and re-emergence of pathogens represent an ongoing, sometimes major, threat to populations. Hantaviruses family Bunyaviridae and their associated human diseases were considered to be confined to Eurasia, but the occurrence of an outbreak in 1993–94 in the southwestern United States led to a great increase in their study among virologists worldwide. Well over 40 hantaviral genotypes have been described, the large majority since 1993, and nearly half of them pathogenic for humans. Hantaviruses cause persistent infections in their reservoir hosts, and in the Americas, human disease is manifest as a cardiopulmonary compromise, hantavirus cardiopulmonary syndrome HCPS , with case-fatality ratios, for the most common viral serotypes, between 30% and 40%. Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity.", "Habitat disturbance and larger-scale ecological disturbances, perhaps including climate change, are among the factors that may have increased the human caseload of HCPS between 1993 and the present. We consider here the features that influence the structure of host population dynamics that may lead to viral outbreaks, as well as the macromolecular determinants of hantaviruses that have been regarded as having potential contribution to pathogenicity. Text: Emerging pathogens cause new or previously unrecognized diseases, and among them, emerging zoonotic diseases are a major concern among scientists studying infectious diseases at different spatial and temporal scales . Changes in biotic and abiotic conditions may alter population disease dynamics and lead to the emergence of zoonotic infections . During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems .", "During the last decades, several outbreaks of emerging and re-emerging viral pathogens have occurred, affecting both purely-local and worldwide/pandemic involvement of human populations. Among the conspicuous examples are influenza A, Ebola virus, hepatitis C virus, severe adult respiratory distress SARS , coronavirus, and human immunodeficiency virus, which challenge prevention and control measures of public health systems . In the Americas, the recent outbreak of pandemic influenza A subtype H1N1 became a major target for control due to its rapid spread, and uncertainties in virulence and transmissibility, yet vaccine availability was limited when significant activity occurred in advance of the traditional influenza season . However, in the last century outbreaks of several viral-related diseases have emerged or re-emerged involving arenaviruses and dengue viruses, and more recently, hantaviruses, and the expansion of the geographic range of West Nile virus. Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 .", "Among zoonotic diseases, small mammals are hosts of several pathogenic RNA viruses, especially Arenaviridae and Bunyaviridae: Hantavirus . Hantavirus infections became a concern in the Americas after the description of an outbreak of acute respiratory distress occurred in the Four Corners area in 1993 . The newly recognized disease, hantavirus cardiopulmonary syndrome, HCPS or hantavirus pulmonary syndrome , was linked to infection by the newly-discovered Sin Nombre virus SNV , and the rodent Peromyscus maniculatus deer mouse was identified as the reservoir . However, hantavirus infections have a much longer history. A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements .", "A review of ancient Chinese writings, dating back to approximately 960 AD, revealed descriptions closely resembling hemorrhagic fever with renal syndrome HFRS , the syndrome caused by Old World hantaviruses . During the twentieth century, cases of acute febrile disease with renal compromise were described from several Eurasian countries and Japan, often in association with military engagements . HFRS as a distinct syndrome, however, was first brought to the attention of western medicine in association with an outbreak that occurred among United Nations troops during the Korean conflict between 1951 and 1954, where more than 3,200 soldiers were afflicted . It took more than two decades until the etiologic agent, Hantaan virus HTNV , was isolated from the striped field mouse Apodemus agrarius, detected in part by the binding of antibodies from patient serum samples to the lung tissues of healthy, wild-caught field mice . The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus .", "The virus was later found to represent the type species of a new genus Hantavirus of the family Bunyaviridae, although it was later apparent that the first hantavirus to be isolated was the shrew-borne Thottapalayam virus . The categorization of hantaviruses as belonging to the family Bunyaviridae is due in part to the consistent presence of three RNA genomes that are circularized in vivo as a result of the presence of terminal complementary nucleotides that help fold the genome into a -hairpin‖ morphology, first described for the Uukuniemi phlebovirus . Table 1 is a list of the predominant, serologically distinct pathogenic hantaviruses. Many other named genotypes are described, but such other pathogenic forms are generally closely related to Andes or, in some cases, Sin Nombre virus. During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, .", "During virus maturation, the precursor form GPC is processed using a membrane -bound protease into Gn and Gc, a cleavage that occurs, and appears to be signaled, after the conserved peptide signal WAASA at the C-terminal of Gn . Although the two proteins can be expressed independently through transfection, they can be retained in the wrong cellular compartment ER or aggresome ; they thus must be co-expressed to allow them stability so that the two can be assembled correctly in the Golgi 25, . A number of activities and properties have been identified for the hantavirus envelope glycoproteins, including some features that are suspected to be involved in the pathogenicity of the disease-causing serotypes, a possibility that has engendered experimental attention. The glycoproteins are the known or presumed ligands for at least two distinct cellular receptors, the 3 integrin chain and decay accelerating factor, or DAF ; with gC1qR/p32 also identified as another potential entry receptor . Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies .", "Comparisons with the tick-borne encephalitis virus E protein, led Tischler et al. to consider the Gc glycoprotein as a potential class II fusion protein, perhaps imparting fusion activity to the virion, and this hypothesis has gained support in other studies . Additional activities have been identified with, or claimed to be related to, Gn. For many of these studies, an underlying premise has held that there are differences between the glycoproteins of -pathogenic‖ hantaviruses relative to viruses in the genus that are dubbed to be -non-pathogenic‖. While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV .", "While it is true that it has not yet been possible to link Prospect Hill virus PHV to human disease, the absence of evidence for its pathogenicity should perhaps not be equated with the evidence of its absence. One might only consider that the level of disease e.g., lethargy, fever, proteinuria, and azotemia associated with infection of nonhuman primates by PHV is not significantly different from that recorded for nonhuman primate models using the known-pathogen Puumala virus PUUV . For the purpose of this discussion we will presume that apathogenic hantaviruses are indeed apathogenic. While some studies have suggested that Gn glycoproteins are directed more rapidly into the ubiquitin-proteosome pathway than are apathogenic forms, others have interpreted differences in the handling of Gn glycoproteins across hantavirus species by the ubiquitin-proteosomal system as independent of pathogenicity . Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host .", "Some investigators have directed their efforts toward identifying a differential capacity, either kinetic or in absolute magnitude, in the ability of pathogenic and apathogenic hantaviruses to elicit an interferon response in cells. One premise that emerges is that apathogenic forms would tend to induce an earlier innate response that would render it more likely that the virus would be quickly cleared or rendered less competent in its replication so as to blunt any pathological response in the host . The anti-hantavirus innate response can in some cases be attributed to viral interaction as a ligand of TLR-3, but not in others, and in endothelial cells, it appears not to require more than the viral particle itself, even when introduced in replication-incompetent form . Proteins and mRNAs prominently induced by hantaviruses include MxA and IFIT-1 ISG-56 and others including some with known or suspected anti-viral activity. Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell .", "Those hantaviruses, often highly pathogenic strains, that fail to induce a potent antiviral response, are suspected or presumed to have a more potent interferon-pathway antagonism mechanism relative to other viruses, a mechanism that acts positively to prevent an effective innate response from forming, at least early in infection . Yet some instances are reported wherein highly pathogenic hantaviruses, such as SNV, are also able to induce expression of interferon-stimulated gene mRNAs, even very early in infection, with ISG proteins, as expected, taking longer to appear in the cell . Anti-interferon activities have also been attributed to the NSs protein that may be elaborated in cells infected by serotypes that encode this protein . Other investigators have examined the activities of hantavirus glycoproteins and other proteins that might themselves directly affect some aspects of the pathogenic progression associated with hantavirus infection of humans, such as vascular permeability changes. While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments.", "While early attempts to directly cause increases in permeability of endothelial monolayers with viral particles or viral infection were largely disappointing, hantaviruses have been identified as adversely affecting endothelial migration over substrata and in potentiating VEG-F-induced endothelial permeability . The shorter 50-kD nucleocapsid or N protein is a structural component of the viral nucleocapsid, along with the genomic viral RNA segments. As an RNA-binding protein that engages the hairpin termini of the genomic segments with high affinity , it limits the access of the RNA to host nucleases and helps to render viral replication a closed process within the cytoplasm. It also acts as a peripheral membrane protein, as does the L protein , an activity that could play a role in its presumed, but not yet demonstrated function as matrix . Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences .", "Until recently, it had not been appreciated that N has a wide variety of other activities, some of which can be linked, not only to fundamental requirements of replication, but also to the interference with an array of the intracellular processes of the normal cell. Thus, an interaction between the amino terminus of the hantavirus N protein and the cellular protein Daxx has been proposed, with the suggestion of potential pro-apoptotic consequences . N is also reported to interact with actin microfilaments, and the SUMO-1 protein . Using reporter-gene based assays, Connie Schmaljohn and her colleagues have reported that Hantaan virus' nucleocapsid protein has an inhibitory role in inflammatory responses mediated by NF kappa B NF-B . The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps.", "The effects on NF-B expression appeared to be confined to prevention of its nuclear translocation after its attempted activation with lipopolysaccharide, LPS . In the cytoplasm of infected cells, N protein can be found in cellular P bodies where it sequesters and protects 5' caps. It may locate the caps through its interaction with DCP1, a key constituent of P bodies. During hantavirus infection, the viral RNAs become concentrated in P bodies, through their interaction with N and DCP1. The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways.", "The N protein demonstrates preferential protection of mRNAs engineered to prematurely terminate their encoded protein in comparison to native mRNAs . N protein has been increasingly linked to viral replication and translation, sometimes in previously unanticipated ways. It is among a growing family of diverse viral proteins that can serve as a nonspecific -RNA chaperone‖, an activity that should facilitate the L polymerase's access to vRNA for transcription and replication, in that it can transiently dissociate misfolded RNA structures . Some of N protein's effects on translation might not immediately be recognized to be adaptive in nature. It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation.", "It can replace the entire EIF4F translational initiation complex, simultaneously presenting the ribosome with a replacement for the cap-binding activity of eIF 4E, binding to the 43S pre-initiation complex as does eIF 4G, while replacing the helicase activity of eIF 4A, which is presumed to be needed to dissociate higher-order RNA structure . These three factors normally work together to achieve translational initiation. In P bodies, N protein's ability to bind at high affinity to capped native cellular oligoribonucleotides, along with its activity in protecting capped RNAs from degradation likely facilitates the access of capped oligonucleotides for use in transcriptional initiation by L polymerase -cap snatching‖ . Trafficking of N for viral assembly: Classically, N protein in infected cells appears to be clustered or particulate in nature, with a heavy concentration at a single perinuclear location, widely considered to be the Golgi . The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster .", "The N proteins of hantaviruses are found in association with particulate fractions, and confocal microscopy and biochemical-inhibitor studies have shown that N tracks along microtubules but not with actin filaments . The ultimate destination for N, for its assembly into viral particles is the Golgi, and it traffics there via the endoplasmic reticulum-Golgi intermediate complex ERGIC , also known as vesicular-tubular cluster . A dominant negative inhibitor, dynamitin, associated with dynein-mediated transport, reduced N's accumulation in the Golgi. Later studies suggested that the specific dependence on microtubular transport is specific to Old World hantaviruses such as HTNV, but that the New World hantavirus ANDV is instead associated with actin filaments . However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness.", "However, recent data indicates that microtubular transport is indeed utilized for the New World hantavirus SNV . Hantavirus diseases of man have long been suspected of having an immunopathogenic basis in part because of their relatively long incubation period of 2-3 weeks and the observed temporal association between immunologic derangements and the first appearance of signs and symptoms of hantavirus illness. HFRS and HCPS share many clinical features, leading many investigators to consider them to be, in essence, different manifestations of a similar pathogenic process, differing mainly in the primary target organs of disease expression Table 2 . The pathogenesis of hantavirus infections is the topic of a continuously-updated review in the series UpToDate . By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis .", "By the time symptoms appear in HCPS, both strong antiviral responses, and, for the more virulent viral genotypes, viral RNA can be detected in blood plasma or nucleated blood cells respectively . At least three studies have correlated plasma viral RNA with disease severity for HCPS and HFRS, suggesting that the replication of the virus plays an ongoing and real-time role in viral pathogenesis . Several hallmark pathologic changes have been identified that occur in both HFRS and HCPS. A critical feature of both is a transient ~ 1-5 days capillary leak involving the kidney and retroperitoneal space in HFRS and the lungs in HCPS. The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses.", "The resulting leakage is exudative in character, with chemical composition high in protein and resembling plasma. The continued experience indicating the strong tissue tropism for endothelial cells, specifically, is among the several factors that make β3 integrin an especially attractive candidate as an important in vivo receptor for hantaviruses. It is likely that hantaviruses arrive at their target tissues through uptake by regional lymph nodes, perhaps with or within an escorting lung histiocyte. The virus seeds local endothelium, where the first few infected cells give rise, ultimately, to a primary viremia, a process that appears to take a long time for hantavirus infections . By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney.", "By the time that secondary viremia emerges, the agents of the more severe forms of HFRS and HCPS have begun to achieve sufficient mass as to induce, through PAMP-PRR interactions and other means, the expression of proinflammatory cytokines . For HCPS, that expression favors the pulmonary bed and lymphoid organs, yet, for unknown reasons, spares the retroperitoneum and, in general, the kidney. In HFRS the situation is reversed, and yet it is often not appreciated that the expected preferential tissue tropism of HFRS-associated viruses and their HCPS-associated counterparts for the renal and pulmonary beds, respectively, is not as one would predict through the manifestations of the two diseases. Local elaboration of inflammatory and chemotactic mediators is considered to be a requirement for the development of systemic disease symptoms, with those abnormalities sometimes culminating in shock and death. Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs.", "Yet it is not hypoxemia, due to the prominent pulmonary edema, that leads to death in most fatal cases of HCPS, but rather intoxication of the heart by as-yet-undefined mediators that leads to the low cardiac output state and the associated shock syndrome . It is tempting to speculate that mediators produced in the lung in connection with the inflammatory infiltrate can percolate through the coronary circulation with minimal dilution in HCPS, a disadvantageous consequence of the close anatomic juxtaposition of the two organs. Thus, at least three classes of potential mechanisms, some overlapping and all certainly nonexclusive of the others, could be presumed to underlie the pathogenesis of HCPS. These include: . Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified.", "Innate immune mechanisms. The nature of interactions between hantavirus pathogen-associated molecular patterns PAMP with the pattern recognition receptors PRR of susceptible endothelial cells are beginning to be clarified. The prototypical HTNV appears to be recognized by TLR-3 . Such an infection has consequences such as increased expression of HLA-DR in dendritic cells and differentiation of monocytes toward dendritic cells . . Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling.", "Direct viral effects. The observed correlation between viral load and disease severity leaves the possibility open that hantavirus particles or RNA can themselves have toxic effects on cells or on signaling. Some investigators have favored direct viral toxicity, acting through the inhibition of endothelial cell barrier function, as an explanation for much of the capillary leak, although there is widespread agreement that multiple mechanisms that mediate pathogenesis likely operate simultaneously in the affected patient . A potentially important clue toward the mechanism by which hantavirus infections deplete blood platelets and, in some cases cause hemorrhagic manifestations, was advanced by the recent discovery that pathogenic hantaviruses are able to recruit platelets to adhere to endothelial cell surfaces, with β3 integrin used as a critical binding element . . Pathogenic effects caused by the activities of specific viral macromolecules.", ". Pathogenic effects caused by the activities of specific viral macromolecules. We have reviewed some of the activities associated with the Gn, Gc and N, virally-encoded polypeptides in previous sections. Testing models of pathogenesis can be done more effectively when there is an animal model that mimics key aspects of the disease. There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences.", "There is no such model that closely mimics HFRS, but animal models exist for both the asymptomatic carriage of PUUV and SNV by their native carrier rodents, the bank vole Myodes glareolus and the deer mouse P. maniculatus; as well as a Syrian hamster model using ANDV or the related Maporal virus from Venezuela, for which an HCPS-mimetic disease is observed . The ANDV-Syrian hamster model has a number of features in common with the human disease, as well as some differences. Unlike the neurologic diseases that have been possible to elicit with HTNV, the hamster model for HCPS appears to be caused by capillary leak that results in pulmonary edema and the production of a pleural effusion with exudative characteristics. Typically the hamsters die between 11 and 14-d post-inoculation, reflecting a slightly accelerated incubation period in comparison to human infections. As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS .", "As with human HCPS, the microscopic examination of the lung reveals abundant fibrin deposition, thickened alveolar septa, and viral antigen expressed abundantly in the microvascular endothelium. ANDV-infected hamsters fitted with physiologic monitoring devices exhibited diminished pulse pressures, tachycardia, and hypotension that appear to closely mimic the shock that is believed to be the proximate cause of demise in patients who succumb to HCPS . Compared to the human disease, ANDV-infected hamsters exhibit exceptionally high titers of live ANDV in their tissues, with much of the viral replication occurring in hepatocytes, which are spared in the human disease. Titers of live ANDV in some cases exceed 10 8 /g, whereas hantavirus isolates from human tissues have been notoriously difficult to obtain. Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus.", "Despite the universal occurrence of mildly-elevated hepatic enzymes in patients with HCPS, hepatic enzymes do not appear to be present at elevated levels in the blood of diseased hamsters even immediately before death . The protracted incubation period associated with hantavirus disease gives the host considerable time to mount a mature immune response against the virus. Thus, in contradistinction to infections of comparable severity and related symptomatology associated with arenaviruses and filoviruses, hantavirus infections of humans are associated with antibody responses of significant titer by the time symptoms commence. Despite this observation, it appears to be possible that natural variation in individual neutralizing antibody responses among patients with SNV infections can be linked to disease severity, suggesting that administration of antiviral antibodies could prove effective therapeutically . In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons.", "In the case of ANDV infection, new evidence has emerged indicating that the apparent clearance of the virus from the blood does not result in the complete removal of antigenic stimulus by the virus, suggesting that the virus may persist, perhaps in some as-yet undetermined immunologically privileged site . A role for T cell-mediated pathological responses in HFRS and HCPS has been the source of speculation for a variety of reasons. The severity of SNV-associated HCPS may have made it more apparent that the onset of pulmonary edema, tachycardia and hypertension seemed to be all but universally temporally associated with the appearance of a spectrum of highly-activated cells of the lymphoid lineage in the peripheral blood. Cells with a close morphologic similarity to these -immunoblasts‖ were detected in the congested, heavy lungs of patients who came to autopsy, as well as in lymphoid organs and in the portal triads 63, . These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, .", "These observations led to speculation that some component of hantavirus pathogenesis could be linked to the appearance of antiviral T cells that could stimulate or contribute to the appearance of a -storm‖ of mediators and the associated capillary leak phenotype. Subsequent studies have borne out the expectation that a significant fraction of the immunoblast population in patients with HCPS are T cells with specificity for specific class I HLA-presented epitopes of viral antigens, including Gn, Gc and N 77, . Presumably, the antiviral activities of such cells, manifested in part through their elaboration of mediators in the affected interstitium, can contribute to the endothelial/capillary leak that lies at the heart of hantavirus pathogenesis. Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. .", "Because early cases of HCPS often came to autopsy, it became possible to examine necropsied tissues for expression of cytokines. The study by Mori et al. . revealed high relative expression of proinflammatory cytokines including TNF, IL-1, IL-6, providing evidence in favor of a -cytokine storm‖ model for pathogenesis . The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well .", "The authors believed, based on the morphology of cytokine-secreting cells, that both monocytes and lymphocytes were contributing to the production of cytokines. That proinflammatory mediators are found in elevated levels in the plasma as well as the renal interstitium of patients with acute hantaviral illness has been recognized for some time as well . While diagnosis of HCPS as well as HFRS is best accomplished with IgM serology, in the acute stage of SNV infection, RT-PCR can also be used if blood cells or blood clot are used instead of plasma or serum, where sensitivity even using nested PCR primers drops to about 70% . In a facility at which many cases of HCPS are treated, the University of New Mexico medical center in Albuquerque, a diagnostic service has long been offered in which the patient's hematologic findings are analyzed to establish the probability that a patient has HCPS. The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents.", "The combination of thrombocytopenia, elevated abundance of -immunoblast‖ lymphocytes, left-shifted polymorphonuclear cell population without strong morphologic evidence for their activation, and elevated hemoglobin or hematocrit values is highly specific for HCPS and allows clinicians the ability to put presumptive-HCPS patients on extracorporeal membrane oxygenation ECMO , which is believed to have saved many patients from a lethal outcome . Human infection by hantaviruses is thought to follow contact with secretions or excretions produced by infected rodents. In the United States, 538 human infections by hantavirus were reported through late December 2009 , with New Mexico, Arizona and Colorado exhibiting the highest case-loads. While the prototypical central American hantavirus in central America was Rio Segundo virus of Reithrodontomys mexicanus from Costa Rica, the first human disease appeared some years later in Panama, where Choclo virus CHOV arose as the etiologic agent and is believed to be responsible for all known cases of HCPS. The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate .", "The fulvous pygmy rice rat Oligoryzomys fulvescens has been identified as the rodent reservoir . In Panama, the first cases of HCPS, albeit with little or no evident cardiac involvement, were reported in 1999, and since then, 106 human infections have occurred with a 26% mortality rate . Serosurveys of mammals in Mexico and Costa Rica have found anti-hantavirus antibodies , and seroprevalences ranging between 0.6 to 1.6% in human populations were reported despite the absence of known HCPS cases . In South America, HCPS cases have been indentified in Argentina, Bolivia, Brazil, Chile, Paraguay and Uruguay, and evidence for human exposure to hantaviruses have also been reported in Venezuela and Perú . In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 .", "In southern South America, ANDV is the main etiologic agent with cases in Chile and Argentina reported since 1995. In Chile, 671 cases of HCPS due to ANDV have occurred during the period 2001-2009 . Since 1995, more than 1,000 HCPS cases have been reported in Argentina ; in Brazil, approximately 1,100 HCPS cases have been identified between 1993 and 2008 . Case-fatality ratios in those three countries have been similar, ranging from 30% Argentina , 36% Chile and 39% Brazil . Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% .", "Hantavirus infections occur more frequently in men than women, although the male/female ratio is highly variable. For example, Panamanian communities showed a ratio of 55 men to 45 women , while in Chile the ratio is more biased to males 71% . In the Paraguayan Chaco the male-female ratio approaches 50% . In North America, by December 2009 63% of case-patients were males . All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized .", "All ethnic and racial groups seem to be susceptible to hantavirus infections, and the differences between certain groups as indigenous and non-indigenous are more likely correlated with the type habitat where the population resides e.g., rural versus urban areas . In fact, rural communities account for the highest hantavirus incidences overall and are therefore at higher risk 92, , although the importance of peridomestic settings as a major area of exposure has also been emphasized . The main mechanism by which humans acquire hantavirus infection is by exposure to aerosols of contaminated rodent feces, urine, and saliva . This can occur when humans reside in areas in close proximity to those that rodents inhabit, live in areas infested with rodents, or when rodents invade human settings, which are more frequent in rural habitats. There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern.", "There is a long history of human co-existence with rodents, raising questions about the apparent recent increases in hantavirus-related illnesses, especially HCPS. Other than an apparent association with El Niño southern oscillation ENSO events in some regions , the recent increases in incidence of HCPS do not seem to follow a readily-defined temporal or spatial pattern. However, some landscape features such as habitat fragmentation or human-disturbed areas may influence rodent population dynamics and impact viral incidence . Despite the stochasticity associated with contraction of hantavirus infection, certain scenarios have been recognized as posing higher risk. Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission .", "Human activities in poorly ventilated buildings that aerosolize particulates that are then inhaled i.e., cleaning, shaking rugs, dusting are frequently identified among patients admitted for HCPS . Outdoor activities are thought to convey lower risk due to lability of hantaviruses to UV radiation and the presumed tendency to be dispersed in wind, although certain environmental conditions seem to maintain the virus for longer periods outside its natural host allowing for indirect transmission . An alternative but uncommon route of virus transmission is by rodent bites . Field workers handling mammals are potentially at higher risk of exposure with hantavirus infections, although when quantified through serosurveys the absolute risk appears rather slight . A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus .", "A new study in Colorado suggests the possibility that a rodent bite may have been the proximate vehicle for outdoor transmission of SNV , which re-emphasizes the use of personal protective equipment during field work activities . As a particular case within hantaviruses, person-to-person transmission has exclusively been documented for the South American Andes virus . The identification of this transmission route has been made using both molecular tools and epidemiological surveys, but the mechanism of interpersonal transmission is not well established. Recent findings show that family clusters and specifically sexual partners share the greater risk of interpersonal transmission, although sexual transmission per se can be neither inferred nor refuted presently . Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions .", "Interestingly, ANDV may also be shed by humans through other biological fluids such as urine , illustrating the particular properties that differentiate this virus from other hantaviruses. Although interpersonal transmission seems to be unique for ANDV, viral RNA of PUUV has been detected in saliva of patients with HFRS, and some patients with SNV-HCPS have viral RNA in tracheal secretions . Hantaviruses in the Americas are naturally hosted by rodents Muridae and Cricetidae as well as shrews Soricidae and moles Talpidae Figure 1 . Three shrew and one mole species have been reported to host hantaviruses and their pathogenicity for humans remains unknown . At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host .", "At least 15 rodent species have been identified as carriers of different pathogenic hantaviruses, with some South American genotypes such as Castelo do Sonhos CDSV or Hu39694 only identified after human infections Figure 1 . Hantaviruses typically show high species-specificity and no intermediate host . However, some hantavirus genotypes have been described in the same rodent species. Such is the case of Playa de Oro OROV and Catacamas CATV identified in Oryzomys couesi , or Maporal MAPV and Choclo CHOV hosted by O. fulvescens . In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis .", "In North America both Muleshoe and Black Creek Canal hantaviruses have been detected in geographically-distant Sigmodon hispidus . Also, one hantavirus genotype e.g., Juquitiba-like virus may be carried by more than one rodent species O. nigripes, Oxymycterus judex, Akodon montesis . Another example is Laguna Negra virus LANV which after being identified in Calomys laucha has also been reported in C. callosus . The rapid increase in the discovery of new hantaviruses and the identification of their hosts does not seem likely to end soon as new small mammal species are screened . This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses .", "This subject is complicated by continued controversy in the criteria for the classification of distinct hantaviruses , which is also tied to host taxonomic classification and taxonomic rearrangements. Cross-species transmission is a major process during spread, emergence, and evolution of RNA viruses . Particularly within hantaviruses, spillover to secondary hosts are increasingly identified as more extensive studies are performed . For example, ANDV is the predominant etiologic agent of HCPS in South America, and O. longicaudatus the main rodent reservoir. Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori .", "Spillover in at least four other rodent species that co-occur with the reservoir have been identified, with Abrothrix longipilis showing the second higher prevalence to ANDV-antibodies, and there is presently no question that the virus is extremely similar genetically between the two host rodents . In North America, spillover of Bayou virus BAYV may have occurred from the main reservoir O. palustris to S. hispidus, R. fulvescens, P. leucopus, and B. taylori . Hantavirus spillover is more likely to occur with host populations inhabiting sympatric or syntopic regions , and cross-species transmission would presumably have greater chances of success if the host species are closely related . An interesting exception is found between Oxbow virus OXBV and Asama virus ASAV in which a host-switch process seemed to have occurred between mammals belonging to two families Talpidae and Soricidae , likely as a result of alternating and recurrent co-divergence of certain taxa through evolutionary time . Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality .", "Hantaviruses are horizontally transmitted between rodents and are not transmitted by arthropods unlike other viruses of the family Bunyaviridae . Spillover infection to nonhuman mammals usually results in no onward or -dead-end‖ transmission, but if humans are infected may result in high morbidity and mortality . During the spring of 1993, an outbreak of patients with HCPS due to SNV occurred in the Four Corners states resulting in more than 60% case-fatality among the initial cases, many involving members of the Navajo tribe . In Panama, an outbreak was reported during 1999-2000 in Los Santos, and 12 cases where identified with three fatalities . This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996.", "This represented the first report of human hantavirus infections in Central America. In South America, the first largest identified outbreak occurred in the Chaco region in northwestern Paraguay during 1995-1996. Seventeen individuals were identified with SNV antibody ELISA or were antigen IHC positive out of 52 suspected cases . Major outbreaks due to ANDV occurred in 1996 in southern Argentina ; in southern Chile clusters of patients presented with hantavirus illness in 1997 . In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model.", "In Brazil, the first outbreak was identified in the Brazilian Amazon Maranhão State in 2000, and involved small villages that resulted in a 13.3% prevalence of those tested 398 total residents . The factors that trigger hantavirus outbreaks are still poorly understood, probably because they result from several interacting biotic and abiotic features whose key parameters are difficult to model. However, the use of new modeling approaches that involve geographical and environmental features seem to be promising in predicting potential hantavirus outbreaks and/or areas of higher risk . Because hantaviruses are known to be directly transmitted from infected to susceptible hosts, the first natural approach is to relate outbreaks to the ecology of the viral hosts. Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found .", "Hantavirus transmission and persistence in rodent populations depends on several factors that interact to affect ecological dynamics of the host, which in turn is strongly influenced by the behavioral characteristics of individual rodent species, to landscape structure, and environmental features . Viral transmission depends on contact rates among susceptible hosts, and despite the prevailing notion that a higher density increases encounters and hence secondary infected hosts, contrasting patterns relating rodent population size and virus prevalence can be found . In addition, it has been shown that SNV transmission follows a contact heterogeneity pattern, where individuals in the population have different probability of transmitting the infection . The understanding of viral transmission proves to be far more complex when species other than the main reservoir host are incorporated in the model. In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure.", "In fact, recent studies have shown that higher hosts species diversity is correlated with lower infection prevalence in North America for P. maniculatus , in Central America for O. fulvescens reservoir of Choclo virus and Zygodontomys brevicauda reservoir of Calabazo virus , and in South America for Akodon montensis reservoir of Jabora virus . Contact rates vary according to the spatial distribution of populations and seem to be strongly influenced by landscape structure. For example, SNV prevalence in P. maniculatus was higher in landscapes with a higher level of fragmentation of the preferred habitat . In addition, certain properties of the landscape such as elevation, slope, and land cover seem to be useful in detecting areas with persistent SNV infections, and therefore thought to be refugial areas where the virus can be maintained for years . Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus .", "Changes in the natural environment of reservoir species, such as forest fragmentation and habitat loss, may alter population abundance and distribution and lead to hantavirus outbreaks, as observed in the Azurero Peninsula of Panama . Also, differences in the microhabitat, including overstory cover, may lead to differences in the ecological dynamics within populations and affect the rate of exposure to the virus . Differences in hantavirus infections through contrasting landscapes in the latitudinal span have been found in rodent populations of O. longicaudatus in Chile, suggesting that humans are differentially exposed to the virus . Rodent population dynamics are affected by seasonal changes of weather and climate . In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño .", "In the case of the ENSO-associated outbreaks, a complex cascade of events triggered by highly unusual rains in the precedent year have been postulated to result in an increase of primary production and rodent densities, also increasing the likelihood of transmission of the virus to humans, but it has proved difficult to precisely demonstrate the suggested intermediate events such as increased rodent densities in the increased caseload . In South America, effects of climate change and hantavirus outbreaks have not been well studied, despite the knowledge that several rodents species that are reservoirs of emerging diseases have dramatically been affected by events like El Niño . Changes in host population dynamics are also affected by seasonality, which may lead to disease outbreaks when processes that equilibrate rodent populations from season to season are interrupted . Viral emergence may continue to be promoted as human-introduced changes continue to increase in the environment at different geographical scales. Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system .", "Human incursions into previously uncultivated environments may lead to new contacts between rodent reservoirs and humans, increasing the likelihood of contracting infections . These changes may also alter rodent's population structure and dynamics and interspecies interactions creating conditions that may lead to viral outbreaks, viral establishment in new hosts, and emergence of HCPS , even with seemingly slight ecological disturbance to the virus-host system . Certain pathophysiologic characteristics, including thrombocytopenia and shock, of hantavirus diseases of humans, bear substantial similarity to the hemorrhagic fevers induced by other viruses such arenaviruses, filoviruses and flaviviruses, despite sharing essentially no sequence similarities therewith. Such observations raise questions about whether such commonalities in pathogenesis are chance similarities of phenotype, or instead report the presence of common molecular mechanisms among the viruses. In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets.", "In this review we discuss the general properties, discoveries and epidemiology/ecology of the New World forms of pathogenic hantaviruses, and also seek to identify some of the characteristics of the viral macromolecules and immunologic mechanisms that have been proposed as potential direct mediators of the pathogenic events that characterize the human disease HCPS. While it is unlikely that expression of any particular viral protein or RNAs in isolation can be relied upon to replicate key phenotypes of infection by the complete virus, some of the findings have been sufficiently consistent with what is known of the pathogenesis in vivo that they offer plausible first-pass leads in the search for therapeutic targets. We look forward to the mechanistic revelations that will follow the inevitably expanded usage of powerful methods such as deep sequencing, ever-more advanced imaging, and microscopic methods, and animal models that can at last be said to be close mimics of human hantavirus disease." ]