mteb/CovidQA huggingface dataset:
For the 143 HMPV genomes, we checked sequence conservation at transcriptional control regions, at the termini of each gene, as well as the lengths of intergenic sequences between gene boundaries. The length of the F-M2 intergenic region was different between group A and B viruses, that is, 13 nt and 2 nt, respectively. The SH-G and G-L intergenic regions were the longest, up to 125 nt and to 190 nt, respectively. Consensus nucleotides (9 to 19 length) at the putative start and end regions flanking the ORF of the viral genes are shown in Fig. 1 . The gene-start and -end regions of N and P were conserved (> 90% average pairwise identity) in both HMPV groups, and the M2 and M gene-start and -end were also conserved in HMPV group A and B, respectively. The putative ATG start codon was consistently located at positions 14-16 upstream of a gene start motif (consensus: GG/AGAC/TAAA/GTnnnnATG), except for the internal M2-2. An additional ATG start codon upstream of the gene-start motif was
observed in the SH gene for the B1 and B2 strains. In five of the eight annotated genes (N, P, F, M2, and G (B1 and B2 strains only)), the intergenic regions were short and the ORFs for these 5 genes terminated within the propositioned gene-end motifs.
We combined the five genome sequences from Kenya and Zambia with available global sequences, aligned individual genes and calculated the percent nucleotide (nt) and amino acid (aa) identity ( Table 2) .
The coding sequences of N, M, F, M2-1, M2-2, and L genes were conserved at nucleotide and amino acid levels, by sharing > 85% between-subgroup nucleotide identity and 90% protein identity ( Table 3 ). The nucleoprotein gene was the most conserved among all subgroups at the nt and aa levels. SH and G glycoprotein genes were more divergent between the HMPV subgroups at the nucleotide level with 76 and 63% identity, respectively. The SH protein length was variable between group A and B strains due to a nucleotide substitution (CAA ➔ TAA) at gene position 532 in group B, resulting in protein lengths of 178 and 180 aa, respectively. The predicted G protein length also varied among the different HMPV subgroups, between 219 and 241 aa, due to different positions of the Stop codon. Amino acid sequence diversity for G and SH glycoproteins is depicted in Fig. 2 and Additional file 2: Figure S2 , respectively. The diversity of the complete nucleotide sequences of SH and G genes is depicted in
phylogenetic trees in Fig. 3 .
We evaluated phylogenetic classification and relationship between the 5 new genomes obtained in this study and previously published genomes (Fig. 3) . Full genome Figure S3 . There was phylogenetic congruence with the individual gene sequence sets as with the full genome dataset, except for F and M2 gene (Additional file 3: Figure S3 ).
Variant or drifted viral strains may lower the sensitivity of detection resulting in a decreased quantitation of the viral load and underestimation of disease incidence [19] . We checked the new HMPV genomes for nucleotide differences in the genomic regions targeted by our diagnostic rRT-PCR primers and probes (Additional file 7: Table S4 ) used for HMPV detection. Up to eight primer-and probetemplate mismatches were identified (Fig. 4) : one mismatch in the forward primer region in HMPV group A (F gene-based rRT-PCR assay, Fig. 4a ); one mismatch in each of the forward and probe target regions in group B (F gene-based rRT-PCR assay, Fig. 4b) ; and 5 different mismatches with the N-gene based rRT-PCR assay (Fig. 4c) . Note, the F gene-based rRT-PCR assays are different or specific to the two HMPV groups.
HMPV causes respiratory illness presenting as mild upper respiratory tract infection or life-threatening severe bronchiolitis and pneumonia primarily in children, sometimes adults as well as immunocompromised individuals [2] . However, HMPV genome sequence data from Africa is sparse and information on genome-wide diversity is limited. In the present study, the whole genome sequences of five HMPV strains from Kenya and Zambia were determined and compared with the genomes published previously from around the world. Comparative sequence analysis indicated fairly conserved positioning of the gene-start and -end regions as well as translational start and -end codons. Variation in genestart and -end sequences can have significant impact on transcription initiation and termination efficiency so that there is more selective pressure preventing changes in these regions [20] , and this likely explains our observation. The additional ATG start codon found upstream of the gene-start motif of the
SH gene was consistent with a previous report [21] , though its role in gene expression is yet to be identified.
These observed sequence conservation in N, M, F, M2-1, M2-2, and L genes is not unusual and is suggestive of functional and structural constraints on diversity, but less expected of the F gene because of its status as a neutralization and protective antigen, similar to its close 'relative' RSV [22] . It has also been suggested that the low diversity in F gene might make a substantial contribution to cross-neutralization and cross-protection between the HMPV subgroups [21] . The relatively high frequency of amino acid diversity in G (and to a lesser extent SH) could be attributable to selective pressure for amino acid change coming from host immunity; and the ability of the protein to tolerate substitutions, which might be due to its proposed extended, unfolded nature [22] . The phylogenetic incongruence observed between whole genome tree and the F and G gene trees, is as reported previously for HMPV [23] , and could be attributed to differential rates of evolution, selection pressure
or past recombination events [24] . The prevalence of HMPV in hospitalized pediatric population in Kilifi county in coastal Kenya has been reported [7, 25] . However, it is notable that in recent years, HMPV has been detected at low prevalence in Kilifi (unpublished observations from hospital-based pneumonia surveillance). Whether this low prevalence is due to reduced virus transmission, or decreased sensitivity of our HMPV molecular diagnostic assay due to progressive primer/probe mismatches, is yet to be established.
We present the first full genome sequences of circulating HMPV strains from sub-Saharan Africa. A limitation of our sequencing method, as is common with amplicon sequencing protocols [26, 27] , was absent coverage at the 3′ leader and 5′ trailer regions not captured by these primers. Our results demonstrate the application of amplicon sequencing to generate full length HMPV genomes directly from clinical samples. The observed diversity of the individual genes is comparable to that described previously [20] [21] [22] . This method and data provide a useful reference for design of local molecular diagnostics and for studies aimed at understanding HMPV epidemiology and evolution in Africa.
Nasopharyngeal and oropharyngeal (NP-OP) swab samples were collected from children (1-59 months) hospitalized with pneumonia, four of whom were enrolled in the PERCH study [18] in 2012. The fifth sample was collected from a child enrolled in the routine pneumonia surveillance study at Kilifi County Hospital, Kenya, in 2015. The samples were tested for HMPV by multiplex semi-quantitative real-time reverse transcription PCR (rRT-PCR) assays. The rRT-PCR primers and probes used, cycling conditions and assay set up have been described elsewhere [28, 29] . Fusion (F) and glycoprotein (G) encoding genes of the HMPV positive samples were amplified in a one-step RT-PCR assay (OneStep RT-PCR kit, QIAGEN), as described previously [7] . Partial G or F nucleotide sequences were analyzed by maximum likelihood (ML) phylogenetic trees using IQ-TREE [30] , together with reference strains of HMPV subgroups (accession numbers AF371337.2, FJ168779, AY297749, AY530095, JN184401 and AY297748). Five HMPV
positive samples from the Kenya and Zambia study sites, belonging to the A2a (n = 1), A2b (n = 2), B1 (n = 1) and B2 (n = 1) genetic subgroups based on their G and F gene sequences, were selected for whole genome sequencing. Data on age, sex and clinical assessment information collected at the time of sample collection, for the five selected samples, are shown in Table 3 .
The sequencing protocol consisted of four steps as follows: (i) primer design, (ii) preparation of primer mixes, (iii) cDNA and PCR (iv) Illumina sequencing and data analysis.
All human metapneumovirus (HMPV) full genome sequences were retrieved from GenBank (January 2018) using the query (txid162145 (Organism) AND 12000(SLEN): 14000(SLEN) NOT patent). Sequence entries with gaps larger than 6 nt were excluded to generate a set of yielding 178 genomes. All possible 23 nt sequences were generated from the genomes dataset and trimmed to a final calculated melting temperature (Tm) of 47.9-49.5°C. Sequences with homology to rRNA sequences, with GC content outside < 0.3 or > 0.75 or with a single nucleotide fractional content of > 0.6 were discarded. The primer set was then made nonredundant yielding 60,746 potential primers. All potential primers were mapped against the 178 HMPV full genomes and the number of perfect matches (frequency score) was determined as a measure of primer sequence conservation. To select primers, the HMPV genome sequences were divided into amplicons with 222 nt overlap spanning the virus genome. Potential primers that mapped within the
terminal 5′ and 3′ 222 nt of each amplicon were identified and the sequence with the highest frequency score was selected, and primers mapping to the reverse bins were reverse complemented. In this manner, 24 primers were selected for each of the 4 HMPV genotype representative genomes (GenBank accession number HMPV A1: AF371337, HMPV A2: FJ168779; HMPV B1: AY525843, and HMPV B2: FJ168778). Because of conservation between genotypes, there was primer redundancy which was removed. The final set of 65 primer sequences, their lengths, calculated Tm, fractional GC content and mapping position on the HMPV genome are presented in Additional file 5: Table S2 . The primers were computationally tested against each of the 4 HMPV subgroups. A graphical representation of the primer target sites is presented in Additional file 1: Figure S1 .
Amplification was performed in two reactions. To avoid generating small products from adjacent forward and reverse primers, amplicons were assigned to alternate Table 3 ).
Bootstrap support values (evaluated by 1000 replicates) are indicated along the branches. Genetic subgroups A1, A2a, A2b, B1, and B2, are indicated. Multiple sequence alignment was done using MAFFT and the ML phylogeny inferred using GTR + Γ nucleotide substitution model and ultrafast bootstrap approximation in IQ-TREE. The genotype B2 Sabana strain sequence (GenBank accession number HM197719) reported from a wild mountain gorilla in Rwanda is marked in blue. The scaled bar indicates nucleotide substitutions per site reactions, with reaction 1 containing primers for amplicons 1,3,5,7,9,11; reaction 2 containing primers for amplicons 2,4,6,8,10,12. Each reverse transcription used Forward Primer Mixes (FPMs) made with 3.0 μl of each reverse primer (100 pmol/μl) plus water to 200 μl to generate a primer concentration of 24 pmol/μl. Two microlitre of the FPM is then used in a 20 μl reverse transcription reaction (2.4 pmol/μl final concentration in reaction or 2.4 μM/primer). For PCR
amplification, each amplicon reaction used a separate PCR Primer Mix (PPM) containing 1.5 μl of each 100 pmol/μl forward primer and 1.5 μl of each reverse primer (5.3-5.5 pmol/μl total primer in the PPM). 2 μl PPM was used per 25 μl PCR reaction = 0.5 pmol/μl in reaction (= 500 nM).
Viral nucleic acids were extracted from the original samples using QIAamp Viral RNA Mini kit (QIAGEN). RNA (5 μl) was reverse transcribed into cDNA using SuperScript III (200 U, Invitrogen), RT buffer (1X final concentration, Invitrogen), and 2 μl of FPM in 20 μl reactions. An aliquot of cDNA (5 μl) was amplified in 35 cycles using Phusion Highfidelity PCR kit (New England Biolabs) and 2 μl of PPM in a 25 μl reaction. The PCR mixture was incubated at 98°C for 30 s, followed by 35 cycles of 98°C for 10 s, 43°C for 30 s, and 72°C for 90s and a final extension of 72°C for 10 min. Expected PCR products for each amplicon were approximately 1500 bp. PCR products from the two reactions for each sample were pooled for Illumina library preparation. Fig. 4 Mismatches between the rRT-PCR diagnostic primers and probes and their expected binding sites in the five genomes from Kenya and Zambia. 'Fwd primer' = Forward primer and 'Rev primer' = Reverse primer. Two rRT-PCR assays were used for HMPV
detection. The colored bars in the figure indicate nucleotide differences (mismatches) between (a) three HMPV-A genomes and HMPV-A specific primers and probes targeting fusion gene, (b) two HMPV-B genomes and HMPV-B specific primers and probes also targeting fusion gene, and (c) all five genomes reported here and specific primers and probes targeting nucleoprotein gene. The sequences of the rRT-PCR primers and probes checked against the African HMPV genomes are listed in Additional file 7: Table S4 Illumina sequencing and data analysis Libraries were prepared using Nextera XT kit (Illumina) and pair-end sequencing (2 × 300 base pairs) with the MiSeq Reagent V3 kit (Illumina), following the manufacturer's instructions. The Nextera enzyme mix was used to simultaneously fragment input DNA and tag with universal adapters in a single tube reaction, followed by 12-cycle PCR reaction for dual indexing. Agencourt AMPure XP beads (Beckman Coulter) were used for all purification steps and
libraries were quantified and quality-checked using the Qubit (Thermo Fisher) and
Bioanalyzer (Agilent). Adapter trimming, quality filtering, kmer normalization of sequencing reads, de novo assembly, calculation of mean genome coverage was as previously described [31] .

A dataset of HMPV genome sequences was retrieved from ViPR in order to infer relationship between HMPV viruses from Kenya and Zambia and viral populations sampled globally. The dataset included 138 sequence entries (> 13,000 nt) that included date (year) and location of sample Table S3 ). Sequence alignment was done using MAFFT v.7.221 [32] using the parameters 'localpair -maxiterate 1000'. IQ-TREE was used to infer maximum likelihood (ML) trees of the complete genome and individual genes under general time-reversible (GTR) substitution model with gamma-distributed among-site rate heterogeneity. A summary of the methodology outlined here is depicted in Fig. 5 .
Viral Respiratory Tract Infections in Adult Patients Attending Outpatient and Emergency Departments, Taiwan, 2012–2013: A PCR/Electrospray Ionization Mass Spectrometry Study

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4635751/

SHA: ef6361c7bffb9e92f397d7004bfb3a9c804d7c6a

Authors: Shih, Hsin-I; Wang, Hsuan-Chen; Su, Ih-Jen; Hsu, Hsiang-Chin; Wang, Jen-Ren; Sun, Hsiao Fang Sunny; Chou, Chien-Hsuan; Ko, Wen-Chien; Hsieh, Ming-I; Wu, Chi-Jung
Date: 2015-09-25
DOI: 10.1097/md.0000000000001545
License: cc-by
Abstract: Viral etiologies of respiratory tract infections (RTIs) have been less studied in adult than in pediatric populations. Furthermore, the ability of PCR/electrospray ionization mass spectrometry (PCR/ESI-MS) to detect enteroviruses and rhinoviruses in respiratory samples has not been well evaluated. We sought to use PCR/ESI-MS to comprehensively investigate the viral epidemiology of adult RTIs, including testing for rhinoviruses and enteroviruses. Nasopharyngeal or throat swabs from 267 adults with acute RTIs (212 upper RTIs and 55 lower RTIs) who visited a local clinic or the outpatient or emergency departments of a medical center in Taiwan between October 2012 and June 2013 were tested for respiratory viruses by both virus isolation and PCR/ESI-MS. Throat swabs from 15 patients with bacterial infections and 27 individuals without active infections were included as control samples. Respiratory viruses were found in 23.6%, 47.2%, and 47.9% of the 267 cases by virus isolation,
PCR/ESI-MS, and both methods, respectively. When both methods were used, the influenza A virus (24.3%) and rhinoviruses (9.4%) were the most frequently identified viruses, whereas human coronaviruses, human metapneumovirus (hMPV), enteroviruses, adenoviruses, respiratory syncytial virus, and parainfluenza viruses were identified in small proportions of cases (<5% of cases for each type of virus). Coinfection was observed in 4.1% of cases. In the control group, only 1 (2.4%) sample tested positive for a respiratory virus by PCR/ESI-MS. Patients who were undergoing steroid treatment, had an active malignancy, or suffered from chronic obstructive pulmonary disease (COPD) were at risk for rhinovirus, hMPV, or parainfluenza infections, respectively. Overall, immunocompromised patients, patients with COPD, and patients receiving dialysis were at risk for noninfluenza respiratory virus infection. Rhinoviruses (12.7%), influenza A virus (10.9%), and parainfluenza viruses (7.3%) were the most
common viruses involved in the 55 cases of lower RTIs. The factors of parainfluenza infection, old age, and immunosuppression were independently associated with lower RTIs. In conclusion, PCR/ESI-MS improved the diagnostic yield for viral RTIs. Non-influenza respiratory virus infections were associated with patients with comorbidities and with lower RTIs. Additional studies that delineate the clinical need for including non-influenza respiratory viruses in the diagnostic work-up in these populations are warranted.
Text: V iral respiratory tract infections (RTIs) in humans occur throughout the year and represent a major cause of clinical visits worldwide. In the past, the viral causes of RTIs were largely unknown, primarily due to the insensitivity of culturebased methods for the detection of viruses or to the narrow spectrum of viral detection using singleplex nucleic acid tests (NATs). Recently, the development of multiplex respiratory NATs has allowed for the simultaneous, rapid, and sensitive detection of multiple viruses, which facilitates comprehensive studies regarding the epidemiology of viral RTIs. Currently, the viral epidemiology of RTIs has been studied more extensively among pediatric populations compared with adult populations throughout the world. 1 Similarly, most studies describing the viral etiology of respiratory illness in Taiwan, a subtropical country in Eastern Asia, were limited to pediatric populations. [2] [3] [4] Thus, studies among adult patients are lacking,
particularly regarding infections due to fastidious or newly identified viruses, such as human metapneumovirus (hMPV) and human coronavirus (hCoV). Overlapping clinical presentations shared by different respiratory viruses make differential diagnoses difficult to perform based solely on the clinical parameters. 5 Moreover, effective antiviral agents are currently restricted to influenza virus infections. Hence, a better understanding of the epidemiology of adult viral RTIs would aid the future design of diagnostic strategies, infection control, and patient management.
Among the various multiplex NATs, multilocus polymerase chain reaction coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) can simultaneously identify and subtype multiple respiratory viruses. [6] [7] [8] [9] Despite the diagnostic potential, the ability of PCR/ESI-MS to detect human enterovirus and rhinovirus in respiratory samples from patients with RTIs has not been well evaluated. Previous PCR/ESI-MS studies in patients with RTIs did not include these 2 viruses in the diagnostic panels. [6] [7] [8] [9] Here, we expanded upon these previous studies utilizing PCR/ESI-MS for respiratory virus detection. We aimed to comprehensively investigate the epidemiology of adult viral RTIs using PCR/ESI-MS and compare the diagnostic performance between PCR/ESI-MS and conventional culture methods for identifying multiple, clinically relevant, respiratory viruses, including enterovirus and rhinovirus.
To conduct a comprehensive epidemiologic study that included patients with and without comorbidity, we enrolled adults (of at least 18 yr of age) with acute RTIs within 7 days of onset who were treated at a local outpatient clinic of YC hospital or the outpatient or emergency departments of National Cheng-Kung University Hospital (NCKUH), a university-affiliated medical center in southern Taiwan, between October 2012 and June 2013. Acute RTI was defined as the simultaneous occurrence of at least 1 respiratory symptom or sign (new or worsening cough, sputum production, sore throat, nasal congestion, rhinorrhea, dyspnea, wheezing, or injected tonsils) and at least 1 of the following symptoms: fever, chills, and cough. Lower RTI (LRTI) was defined as the presence of acute RTI and a new infiltrate on chest radiograph. For patients experiencing more than 1 episode of RTI, the most recent episode was counted as separate only if the patient fully recovered from the previous episode and there
was a least a 3-week interval between the onset of the 2 episodes. Clinical, laboratory, and radiological data and the contact history of each patient were retrieved. Comorbidities were assessed in all patients based on the Charlson comorbidity index (CCI). 10 Steroid use was defined as the receipt of corticosteroid treatment (10 mg prednisolone or an equivalent daily dosage) for more than 2 weeks. An immunocompromised state was diagnosed if the patients met one of the following conditions: corticosteroid treatment, solid organ or hematopoietic stem cell recipient, or chemotherapy for an underlying malignancy during the past 6 months.
Nasopharyngeal or throat swabs were obtained from all patients and collected in transport medium, as previously described. 11 for virus detection and identification by both virus isolation and PCR/ESI-MS. Clinical specimens were stored at 48C and transported to the study sites within 24 hours of collection. Throat swabs from 42 cases without respiratory infections during the month prior to enrollment were included as control samples for PCR/ESI-MS analysis, including 15 patients with exclusively bacterial infections (documented cases of bacteremia or urinary tract infection) who were admitted to NCKUH and 27 individuals without active infections. These subjects without active infections included 10 patients with stable chronic diseases followed up in NCKUH clinics and 17 healthy individuals whose medical information was collected using a clinical questionnaire.
The study was approved by the Institutional Review Board (B-ER-101-031) of the study hospital, and all patients provided informed consent.
Respiratory specimens were inoculated onto appropriate tissue cultures (Madin-Darby canine kidney, MRC-5, A549, and rhabdomyosarcoma) to isolate human influenza virus, parainfluenza virus, genus Enterovirus, cytomegalovirus (CMV), adenovirus, respiratory syncytial virus (RSV), herpes simplex viruses 1 and 2 (HSV-1 and -2), and varicella zoster virus (VZV). The isolation and identification of viruses were performed using a previously described method 11 and enteroviruses were identified by a immunofluorescence assay using a Chemicon Pan EV mix that cross-reacts with rhinovirus (Light Diagnostics, Chemicon [Millipore], MA). 11, 12 Virus Detection and Identification by PCR/ESI-MS Total nucleic acids were extracted from 700 mL of swab samples using a nucleic acid autoextractor (MagNA Pure Compact Instrument, Mannheim, Germany), and the eluate was stored at À808C until analysis. During the analyses, the extracted nucleic acids were added to both a PLEX-ID Respiratory Virus assay plate and