Molecular characterization of human Respiratory Syncytial Virus in Mexico (season 2023–2024) through whole-genome sequencing

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Genomic surveillance of hRSV is currently of interest for understanding of viral evolution and the monitoring of genetic variations that may affect transmissibility and pathogenicity. Herein, we sequenced complete genomes of hRSV-A and B from season 2023–2024, isolated from pediatric and adult patients with SARI. Methods One hundred pediatrics and 43 adult hospitalized patients, as well as 14 non-hospitalized adult patients positive to hRSV were enrolled. Libraries of hRSV complete genome were generated and sequenced on a MiSeq platform. Phylogenetic analysis and maximum likelihood trees were constructed with the 81 hRSV A and 29 hRSV B sequences obtained in our study. Additionally, we analyzed the list of non-synonymous substitutions and their frequencies for each of the eleven viral proteins. Results hRSV A was prevalent (68%) and children under five years old was the principal group affected. The hRSV A isolates belonged to the A.D lineage and sub-lineages A.D.1.5, A.D.1.8, A.D.3, and A.D.5.2 were prevalent. The hRSV B subgroup was less diverse since the dominant sub-lineage was B.D.E.1. Amino acid substitutions per viral isolate for each of the eleven viral proteins indicated higher variability in hRSV A compared to hRSV B. As expected, we observed a high diversity of substitutions in proteins G, F and L. Conclusions Several lineages and high rate of mutation mainly in RSV-A were found during winter season 2023–2024 in Mexico. The increasing availability of complete hRSV genome sequences will facilitate the surveillance of specific substitutions, thereby contributing to a better understanding of viral evolution and the effectiveness of prophylactic strategies. Biological sciences/Microbiology/Virology Biological sciences/Immunology/Infectious diseases/Viral infection Human respiratory syncytial virus whole-genome sequencing phylogeny lineages Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Human respiratory syncytial virus (hRSV) is an enveloped virus from the Pneumoviridae family, which is a leading etiology of acute lower respiratory tract infection (ALRTI) in children under five years of age. Annually, hRSV produces approximately 33.0 million ALRTI, along with 3.6 million hospitalizations and 26,300 in-hospital deaths ( 1 ). In adults over 65 years of age hRSV-related hospitalizations are estimated from 356,000 to 466,000 in industrialized countries and up to 33,000 in-hospital deaths ( 2 ). The hRSV genome consists of ten genes encoded in a single-stranded RNA of approximately 15,200 nucleotides (nt) in length that expresses eleven proteins: NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2 and L. The envelope glycoproteins G and F mediate viral attachment and entry to the host cells, respectively, and are major targets of the immune response. The G protein is expressed as a precursor of 32 kDa that is modified by N- and O-glycosylation becoming a mature protein of 80–90 kDa ( 3 ). The heavily glycosylated sequence is arranged as an ectodomain comprising two highly variable mucin-like domains connected by a central conserved region (aa 163–189) that includes a highly conserved sequence (aa 164–176), and four cysteines maintained in all viral strains. Additionally, this central region contains a CX3C motif (aa 182–186), which binds to both the fractalkine receptor to induce leukocyte chemotaxis and an heparan binding sequence (aa 184–197) ( 4 )( 5 ). The F glycoprotein is synthesized as a precursor, F(0), which is cleaved by a furin-like protease into two subunits, F1 and F2, with the release of a 27-amino acid peptide. F1 and F2 remain linked by two disulfide bridges. F1 contains the fusion peptide that mediates virus entry through fusion of its envelope with the cellular membrane, following the interaction with the viral receptor ( 6 ). Therefore, F exists in a metastable prefusion conformation and a highly stable postfusion structure. Six antigenic sites have been identified in F (denoted as ø–V), of which sites ø and V are exclusively found in the prefusion state ( 3 ). Interestingly, human neutralizing antibodies with high potency target the prefusion antigenic sites. As a result, the recently approved prophylactic monoclonal antibody Nirsevimab, as well as the vaccines Abrysvo and Arexvy, were designed to target the prefusion conformation of F ( 7 ). Two antigenic subgroups of hRSV, A and B, are recognized based on cross-neutralization assays and the sequence variation of the G gene, which exhibits the highest variability among the viral genes ( 8 ). Since its first isolation in 1956, this virus has undergone genetic diversification, leading to the identification of emergent genotypes and lineages. Particularly, it was identified in the period of June to August 1999 in Buenos Aires, Argentina, a 60-nt duplication (20 amino acid) in the G gene of hRSV B (Trento et al., 2003, DOI 10.1099/vir.0.19357-0 ). Also, in season 2010–2011 in Ontario, Canada, it was observed a duplication of 72-nt (24 amino acid) in the G gene of a hRSV A isolate ( 9 ). Currently, both variants are globally dominant and continue to experience genomic changes. Surveillance of hRSV whole-genome has gained relevance in the context of the newly approved prophylactic therapies to understand virus evolution and evaluate the potential emergence of immune escape variants. Standardized criteria for the classification and genotyping of hRSV are essential in molecular epidemiology for monitoring the circulation of variants globally. Recently, Goya et al., proposed a phylogenetic classification using whole-hRSV genome sequences and a system based on amino acid markers to define distinct lineages ( 10 ). Herein we describe the molecular characteristics of the hRSV circulating in Mexico City during the season 2023–2024. Methods Sample Selection As part of a surveillance program at the Instituto Nacional de Enfermedades Respiratorias (INER), nasopharyngeal swabs of individuals presenting with Case of Influenza-Like Illness (ILI) and severe acute respiratory infection (SARI) are routinely collected. Patients with acute respiratory disease or chronic lung disease exacerbation requiring hospitalization, as well as ambulatory cases are sampled to be tested. One hundred and forty-three hospitalized patients, of which 100 were children and 43 adults, as well as 14 non-hospitalized adult patients were enrolled in this study. All patients were confirmed by the RT-qPCR diagnostic kit Respiratory Panel Filmarray (BioMérieux, Spain). hRSV subgroup identification (subtyping) and cycle threshold (Ct) values were determined by RT-qPCR, using in-house designed primers for detection of the nucleocapsid (N) gene ( 11 ). Viral RNA extraction and whole genome amplification Whole genome sequencing was performed in samples with a high viral load (Ct<25) to facilitate amplification and sequencing. Viral RNA was extracted from 200 µl of nasal swabs in viral transport media (VTM), using QIAamp Viral RNA mini kit (QIAGEN). The whole genome was amplified simultaneously and directly from clinical samples, using panel of primers modified and optimized for multiplex PCR ( 12 ). These primers were used to cover the complete hRSV genome (both A and B) by splitting into two pools of non-consecutive amplicons. An alternative method was used for hRSV-A positive samples from winter season 2022–2023, alternative methodology was used ( 11 ). For the hRSV-B samples COI-5 and COI-7, which were identified as co-infections by initial PCR testing, the VirCapSeq-VERT (VCS) protocol was utilized to sequence the complete genomes of all vertebrate viruses ( 13 ). VCS data were analyzed with the Rapid Identification of Microbes (RIM), a custom viral metagenomics pipeline ( 14 ). Libraries preparation and Sequencing Libraries of hRSV complete genome were generated using the reagents of the Covid-Seq kit (Illumina, San Diego, CA, USA). Libraries were sequenced on a MiSeq sequencing platform using a 2 x 150-cycle to obtain paired-end reads (Illumina, San Diego, CA, USA). The DRAGEN COVIDSeq Targeted Microbial Pipeline on BaseSpace Sequence Hub performed the analysis, mapping, and consensus. For COI-5 and COI-7, libraries were prepared with Twist library preparation and fast hybridization reagents (Twist Bioscience, San Francisco, CA, USA), and sequenced on a NextSeq 2000 instrument using a 2 x 150-cycle to obtain single-end reads (Illumina, San Diego, CA, USA). Mapping and consensus were carried out with the RIM pipeline. Phylogenetic and mutations analysis To perform phylogenetic analysis, we analyzed for hRSV-A 1,032 complete genomes and selected 877 sequences of different states in the USA from 2022 to 2024, 25 sequences of Panama from 2022–2023 and 49 sequences of Canada, available on the GISAID platform. We analyze the 81 whole genome sequences of this study, including 19 sequences from winter season 2022–2023. For hRSV-B, 506 complete genomes 436 sequences of different states in the USA from 2021 to 2024 and 41 from Nicaragua, available on the GISAID platform. We included the 29 whole genome sequences from this study. A maximum likelihood tree was constructed for the whole genome sequence using MEGA 10.0. The General Time-Reversible model was selected with five-parameter gamma-distributed rates and 1000 bootstrap replicates. Edition of the trees was made using FigTree ( 15 ). hRSV-A and hRSV-B clades were assigned by using the real-time phylogenetic analysis in Nextclade ( 16 ). Non-synonymous substitutions were obtained using hRSV-A and hRSV-B reference sequence A/England/397/2017 accession number EPI_ISL_412866 and B/Australia/VIC-RCH056/2019 accession number EPI_ISL_1653999 respectively, in Nextclade ( 17 ) Results During the 2023–2024 season, a total of 100 pediatric patients (aged 1 month to 17 years) and 43 adult patients (mean age 55 ± 18 years) with confirmed hRSV infection by RT-qPCR were hospitalized at the INER. Comorbidities were identified in 68% of children, with the most common being malnutrition (18%), followed by recurrent wheezing (14%), gastroesophageal reflux diseases (12%), asthma (11%) and allergic rhinitis (11%) (Table 1). Subgroup identification was performed in 88/100 pediatric samples, of which 68% were positive for hRSV A and 20% positive for hRSV B. Additionally, 13% of pediatric samples tested positive for hRSV by Viral Respiratory Panel (Filmarray) but could not be further subtyped by the specific RT-qPCR assay. In hospitalized adults, the most common comorbidities were chronic lung disease, asthma, COPD, IFD, obesity and type 2 diabetes. hRSV phylogeny Whole hRSV genome sequencing was achieved of 70 respiratory samples from pediatrics and 40 from adults (Table 2), of which, 81 correspond to hRSV-A and 29 to hRSV-B. These sequences included; 14 of non-hospitalized adult patients from winter season 2023-2024 and 19 from winter season 2022-2023 (GISAID accession number EPI_ISL_19500979- EPI_ISL_19501009 and EPI_ISL_19504707-EPI_ISL_19504788). Phylogenetic analysis indicated that all hRSV-A isolates belonged to the A.D lineage (which carry the duplication of 72 nt in the G gene) and ten sublineages (Fig 1). The sublineages A.D.1.5, A.D.1.8, A.D.3, and A.D.5.2 were prevalent (66/81 isolates, Table 3), mainly in children under 5 years old. The analysis using complete genome showed that Mexican sequences collected in winter season 2023-2024 formed 3 different groups: A.D.1 and sub-lineages; A.D.3 and sub-lineages, and A.D.5 and sub-linages (Fig 1). Remarkably, two sequences of 2023 belong to clade A.D. which is the ancestor of the newest sub-lineages. Each group clustered with sequences from USA from 2022-2024, 2023-2024 and 2022-2024 respectively (Fig 1). The hRSV-B sequences were less diverse (Fig 2), with all belonging to the B.D lineage (characterized by the 60 nt duplication in the G gene) whereas the dominant sub-lineage was B.D.E.1 (28/29 isolates). Mexican sequences (B.D.E.1 lineage) clustered with 2022-2024 sequences from USA (Fig 2). Lineage-defining amino acids in Mexican sequences were present mainly in G, L and F genes and are summarized in Table 4. Amino acid substitutions in hRSV proteins The total number of amino acid substitutions per hRSV isolate is shown in the heat maps in Figure 3. Among the 81 hRSV-A sequenced samples, it was found a median value of 43 amino acid substitutions per viral genome (range 23–88, Fig. 3A), while for the 29 hRSV B positive samples the median value was of 31 substitutions per viral genome (range 0–60, Fig. 3B). Analysis between age groups showed a significantly increased number of substitutions in hRSV-A isolates from younger individuals, 0–5 years old, with respect to the 22–49 years old group. In contrast, the total number of substitutions in hRSV-B was not significantly different between either age group (Fig. 3C). The highest variability was observed in the G glycoprotein (Fig. 3A and B) from both hRSV subgroups. Table 5 summarizes the total number of all the diverse substitutions identified in the eleven encoded proteins from virus isolates. Although the highest number of substitutions was identified in the L protein, the substitution density, which is related to the length of each protein, was higher in the protein G, followed by protein M2-2 in hRSV-A and SH in hRSV-B. Interestingly, hRSV-A showed more variation than hRSV-B, as the calculated hRSV-A/hRSV-B substitution ratio was >1 for each protein, excepting for the M protein with ratio of 0.8. Several amino acid substitutions were exclusive to individual genome and all of them are reported in Additional_file_1. Figures 4 and 5 show amino acid substitutions identified in at least two viral genomes, particularly in proteins NS2, N, P, M, SH, M2-1 and M2-2. The lowest number and frequency of mutations was identified in the protein NS1 from hRSV-A and the phosphoprotein P from hRSV-B (Additional_file_1). In contrast, mutations with frequencies >40% were detected in proteins N, P, M, M2-1, and M2-2 of hRSV-A, as well as in proteins N and M2-2 of hRSV-B (Fig. 4 and 5). Substitutions in the envelope glycoproteins that mediate viral entry and the polymerase were diverse and many of those displayed frequencies higher than 50% (Figure 6), except in the F protein of hRSV-A. More frequent amino acid substitutions in the glycoprotein F of hRSV-A were localized in the signal region (T12I, T13A, L15F), F2 (I59V, A103T), p27 (L119F, T122A, N124I, V127I), as well as in the ectodomain (K470R, A518V) and the cytoplasmic region (R553K) of F1 (Fig. 6A). Low frequency substitutions were identified in the fusion peptide (S146P, I148S, V154I), the antigenic site ø (I206A), the antigenic site II, specifically in the binding site of Palivizumab (L258I, N262K and S276N) and the transmembrane region (V536K). The F protein of hRSV-B showed less variability than that of hRSV-A, although some substitutions were detected with frequencies close to 100%. Of those, three were localized to the ectodomain of F1 (S190N, S211N, S389P) and particularly, S211N was localized in the antigenic site ø (Fig. 7A). Other two low frequency substitutions in site ø were M206I and R209Q. In the glycoprotein G, most substitutions were localized, as expected, to the mucin-like regions 1 and 2. Interestingly, G from hRSV-A showed three substitutions in the central conserved region (F168Y, S177G, N178G) of which, F168Y was within the highly conserved domain (HCD, Fig. 6B). In hRSV-B the substitution T198I with frequency of 10% was also localized in the central conserved region (Fig. 7B). Finally, the viral polymerase displayed most substitutions in the RdRp domain of both hRSV-A and hRSV-B (102 and 36 substitutions, respectively), whereas the less variable region was the catalytic domain with MTase activity (Fig 6C and 7C; Supplementary material). Positions R1339 and G1855 have been described relevant for capping and MTase activity, respectively (18), and we identified substitutions R1339P and G1855R in our hRSV-A isolates (Additional_file_1). Discussion Hospitalizations due to acute hRSV lower respiratory tract infections during the 2023–2024 season showed that infants under five years of age were the primary affected group. hRSV-A exhibited a higher prevalence compared to hRSV B in both pediatric and adult patients. Particularly, infections by hRSV-A outnumbered those by hRSV-B by a factor of 3.4 in pediatric patients and approximately 2.1 in adults (Tables 1 and 2). Previous studies have documented that hRSV-A replicates faster and at higher titers (up to 2 log 10 PFU/ml) than hRSV B in airway human epithelial cell cultures (19) (20). However, similar viral loads have been found in respiratory samples from infants infected with either subgroup A or B, as measured by RT-PCR or viral titration (21) (22), suggesting that factors besides replication rate contribute to the higher prevalence of hRSV-A. Our phylogenetic analysis showed that the prevalent lineages of hRSV-A were A.D.1.5, A.D.1.8, A.D.3, and A.D.5.2, while for hRSV-B, the predominant lineage was B.D.E.1. Studies conducted in three different countries reported the prevalence of the following clades after season 2021–2022: A.D.3, A.D.5, A.D.5.2 and B.D.E.1 in USA (Minnesota); A.D.3, A.D.5.2, B.D.4.1.1 and B.D.E.1 in China (Beijing); and A.D.1, A.D.3.1, A.D.5.2 and B.D.E.1 in Italy (Sicily) (23) (24) (25). Accordingly, A.D.3, A.D.5.2 and B.D.E.1 may have global distribution (25). Notably, the study from Beijing, China reported that B.D.E.1 became prevalent since November 2023 and affected a significantly higher proportion of patients ≥60 years (3-fold increase) than did its parent lineage B.D.4.1. However, B.D.E.1 was mostly associated with development of upper respiratory tract infections and non-severe pneumonia, conversely to the severe diseases caused by the previously dominant lineages A.D, A.D.3 and B.D.4.1 (25). Amino acid substitutions per viral genome and the substitution ratio (hRSV-A/hRSV-B) for each protein indicated higher variability in hRSV-A compared to hRSV-B. Total substitutions were significantly higher for hRSV-A infecting the 0–5-year-old age group than in the 22–49-year-old group. This difference may be at least partially explained by the prevailing circulation of the hRSV-A in children, a host population with a naïve or minimally experienced immune system. This condition facilitates higher viral replication, which in turn can lead to an increased mutation rate (26) (27). As expected, we observed a high diversity of substitutions in proteins G, F and L. However, all substitutions in the F protein showed frequencies <40%, whereas many substitutions in proteins G and L displayed frequencies of up to100%. The F protein contains six potential N-glycosylation sites located at positions N27, N70, N116, N120, N126 and N500. N116 and N126 are within the p27 fragment, which is cleavaged by furin-like cellular proteases (28), while N120 is not conserved among different hRSV isolates (29). Both, the double mutant N27Q/N70Q and the individual N500Q impair membrane fusion (29). We did not identify substitutions in the N-glycosylation sites of our viral isolates. However, we observed the substitution T122A (with a frequency of 28.2%), located in the consensus N-glycosylation sequence N-X-T/S. It has been suggested that T122A might reduce the glycosylation at N120, although this likely has no significant impact on fusion activity (29). On the other hand, we also detected substitutions in the F protein located within the antigenic sites ø and II. Particularly, the low frequency substitutions L258I and N262K were found in the Palivizumab binding site (residues 258 to 275) of hRSV-A, along with the adjacent substitution S276N. Zhu et al., reported 46 polymorphic sites in the extracellular region of the F protein of clinical isolates from children without prophylactic treatment, (including the variation at position 276 (30). Using microneutralization assays, they determined that only the substitutions N262D and S275F conferred resistance to Palivizumab. These substitutions were identified in 2 of 145 hRSV A isolates by the authors and were considered as natural polymorphisms. In our study, the substitution N262K was identified only in 1 of 81 hRSV-A isolates. Palivizumab resistance has been observed in 5–10% of immunocompromised infants with long-term infections and under treatment with Palivizumab. In such cases, substitutions N262D, K272E and S276N have been reported, although only the first two were associated with Palivizumab resistance (31) (30). The recently approved prophylactic monoclonal antibody Nirsevimab targets a prefusion discontinuous neutralizing epitope within site Ø, spanning residues 62 to 69 and 196 to 212 (32). Although most residues within the binding site are conserved at a frequency of >99%, a partial reduction in susceptibility to Nirsevimab has been associated with specific substitutions. These include N208D/S, K65Q/T and the dual substitution N67I/N208Y in hRSV-A, or dual substitutions K68N/N201S and K65Q/S211N in hRSV-B (33) (34). In our study, we identified the substitution I206A (3.8%) in hRSV-A, and the substitutions M206I (3.4%), R209Q (6.9%), and S211N (96.6%) in hRSV-B. These substitutions within the Nirsevimab binding site have been documented as natural polymorphisms, and through microneutralization assays it has been determined that changes in the position 206 of the hRSV A-F protein do not modify the neutralization activity of Nirsevimab, while single mutations in positions 206 and 209 only partially reduce neutralization activity against hRSV-B isolates (34). Other substitutions in F, localized in the signal peptide, p27, transmembrane and cytoplasmic domains showed frequencies >13%. Coincident with our observations it has been previously reported that domains with the greatest number of non-synonymous changes and amino acid positions with higher entropy values are within the signal peptide, p27, heptad repeat domain 2, antigenic site ø, and the transmembrane domain (35). Given the high variability of the antigenic site ø (mainly in hRSV-B), it is of relevance a continued surveillance of emerging variants besides the recently approved anti-hRSV vaccines and the monoclonal antibody Nirsevimab (36). As predicted, the G glycoprotein showed high variability, primarily in the second mucin-like region. However, we also identified the substitutions F168Y, S177G and N178G in the central conserved domain (CCD) of hRSV-A and substitution T198I in hRSV-B. Attachment of hRSV to primary respiratory epithelial cells occurs by binding of the G protein to CX3CR1 and infection is attenuated in viruses lacking the G protein or with mutations in the CX3C motif (37). Although the highly glycosylated domains of the G protein are poor immunogens, antibodies against epitopes within the CCD have been detected. These antibodies can induce antibody-dependent cellular cytotoxicity or block the CX3C–CX3CR1 interaction (38) (39). Furthermore, substitution as 177Q and 177R in the CCD enhance G protein immunogenicity and induce IgG antibodies that inhibit the CX3C–CX3CR1 interaction, thereby reducing pulmonary cell infiltration and lung damage in mice (40). In our study, we identified the substitution S177G at a low frequency (3.7%) in hRSV-A isolates. It would be of interest to evaluate if the serum from individuals infected with this variant can reduce hRSV infection of primary airway epithelial cells. On the other hand, Li et al., previously reported that substitutions T113I, V131D, N178 G, H258Q and H266 L in the G protein are associated with decreased disease severity in hospitalized infants (41). We found the same five substitutions in hRSV-A isolates, with frequencies of 13.6% to 17.3%. Further studies are necessary to assess the impact of these substitutions on viral infectivity. Regarding the L protein or RNA-dependent RNA polymerase, we identified 308 and 85 different non-synonymous substitutions in hRSV-A and hRSV-B isolates, respectively. This enzyme is multifunctional, as it not only participates in the transcription and replication of the viral genome but also exhibits polyribonucleotidyltransferase (PRNTase) activity to add the cap structure, as well as methylase activity to methylate the cap (42). Certain mutations in the L protein have been identified in the context of studies with antiviral drugs that inhibit its enzymatic activity (43) (44) (45). In our analysis, we did not identify substitutions associated with antiviral drug resistance. However, we observed substitutions R1339P and G1855R within the catalytic pocket of the capping domain and the SAM/SAH GxGxGx binding motif of the MTase domain, respectively (18). Both substitutions were present at a frequency of 1.2%. Substitutions in hRSV proteins other than F, G and L have been less extensively studied. Nevertheless, the growing availability of complete hRSV genome sequences will facilitate the surveillance of specific substitutions and their frequencies, thereby contributing to a better understanding of viral evolution and the effectiveness of prevention and treatment strategies. Declarations Acknowledgments We thank Eduardo Márquez García from the Unidad de Biología Molecular, INER for technical assistance in Illumina sequencing. Also, to Carlos Santiago-Olivares and Ana Flisser for technical assistance and provide equipment and materials respectively. We also thank all physicians in the ICU for assistance with patient management. Authors´contributions ERT, JRP, JKW and JAVP conceived and designed the project. VHAT, MCL, FBPO, and MASL collected the clinical data and constructed the database. ERT, FMN, AVJ, EMR, JPC and JVP performed the experimental laboratory procedures. ERT, XJL and EMR performed the bioinformatics and statistical analyses. EBV, FBPO, GB, JDCA, EGC and CGG performed the interpretation of clinical data. ERT, JKW, VHAT, and JAVP wrote the manuscript. JAVP supervised the project and led the team. All authors have read and agreed to the published version of the manuscript. Availability of data and material The genomic information generated during the current study is available in GISAID database. Sequences of RSV-A and RSV-B from Mexico were deposited in GISAID under accession number EPI_ISL_19500979- EPI_ISL_19501009 and EPI_ISL_19504707-EPI_ISL_19504788. Competing Interests The authors declare that they have no competing interests. The sponsors had no role in the design, execution, interpretation, or writing of the study. Ethics approval and consent to participate Institutional Review Board Statement: This study was reviewed and approved by the Science, Biosecurity, and Bioethics Committee of the Instituto Nacional de Enfermedades Respiratorias (protocol number B22-23). The research carried out on humans and/or human data is in compliance with the Helsinki Declaration. Written informed consent was obtained from the patients and/or from their relatives or authorized legal guardians prior to the publication of this paper. Funding This study was financially supported by the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT), Mexico, project CBF2023-2024-2356. This work was financially supported also by Direccion General de Politicas de Investigacion en Salud (DGPIS), Grant “FPIS2024-INER-4886” to J.A.V.-P. The use of VirCapSeq-VERT and related work at Columbia University’s Global Alliance for Preventing Pandemics (GAPP) were supported by the Skoll Foundation (J.K.W., J.P.C., and X.J.L.). 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Novel imported clades accelerated the RSV surge in Beijing, China, 2023–2024. J. Infect. 89 (6), 106321 (2024). Elena, S. F. & Sanjuán, R. Adaptive value of high mutation rates of RNA viruses: separating causes from consequences. J. Virol. 79 (18), 11555–11558 (2005). Regoes, R. R., Hamblin, S. & Tanaka, M. M. Viral mutation rates: modelling the roles of within-host viral dynamics and the trade-off between replication fidelity and speed. Proc. Biol. Sci. 280 (1750), 20122047 (2013). Leemans, A. et al. Characterization of the role of N-glycosylation sites in the respiratory syncytial virus fusion protein in virus replication, syncytium formation and antigenicity. Virus Res. 266 , 58–68 (2019). Zimmer, G., Trotz, I. & Herrler, G. N-glycans of F protein differentially affect fusion activity of human respiratory syncytial virus. J. Virol. 75 (10), 4744–4751 (2001). Zhu, Q. et al. 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Sequence variability of the respiratory syncytial virus (RSV) fusion gene among contemporary and historical genotypes of RSV/A and RSV/B. PLoS One . 12 (4), e0175792 (2017). Nuttens, C. et al. Differences Between RSV A and RSV B Subgroups and Implications for Pharmaceutical Preventive Measures. Infect. Dis. Ther. 13 (8), 1725–1742 (2024). Johnson, S. M. et al. Respiratory Syncytial Virus Uses CX3CR1 as a Receptor on Primary Human Airway Epithelial Cultures. PLoS Pathog . 11 (12), e1005318 (2015). Cortjens, B. et al. Broadly Reactive Anti-Respiratory Syncytial Virus G Antibodies from Exposed Individuals Effectively Inhibit Infection of Primary Airway Epithelial Cells. J. Virol. 91 (10), e02357–e02316 (2017). Bergeron, H. C. et al. Immunogenicity and protective efficacy of an RSV G S177Q central conserved domain nanoparticle vaccine. Front. Immunol. 14 , 1215323 (2023). Bergeron, H. C., Murray, J., Nuñez Castrejon, A. M., DuBois, R. M. & Tripp, R. A. Respiratory Syncytial Virus (RSV) G Protein Vaccines With Central Conserved Domain Mutations Induce CX3C-CX3CR1 Blocking Antibodies. Viruses 13 (2), 352 (2021). Li, W. et al. Disease severity of respiratory syncytial virus (RSV) infection correlate to a novel set of five amino acid substitutions in the RSV attachment glycoprotein (G) in China. Virus Res. 281 , 197937 (2020). Morin, B., Kranzusch, P. J., Rahmeh, A. A. & Whelan, S. P. J. The polymerase of negative-stranded RNA viruses. Curr. Opin. Virol. 3 (2), 103–110 (2013). Tiong-Yip, C. L. et al. Characterization of a respiratory syncytial virus L protein inhibitor. Antimicrob. Agents Chemother. 58 (7), 3867–3873 (2014). Yu, X. et al. Structural and mechanistic insights into the inhibition of respiratory syncytial virus polymerase by a non-nucleoside inhibitor. Commun. Biol. 6 (1), 1074 (2023). Atchison, E. B. et al. Interaction Between the Matrix Protein and the Polymerase Complex of Respiratory Syncytial Virus. Viruses 16 (12), 1881 (2024). Tables Tables 1 to 5 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.xlsx Tables.docx Cite Share Download PDF Status: Published Journal Publication published 28 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 26 Mar, 2025 Reviews received at journal 23 Mar, 2025 Reviews received at journal 14 Mar, 2025 Reviews received at journal 06 Mar, 2025 Reviewers agreed at journal 04 Mar, 2025 Reviewers agreed at journal 25 Feb, 2025 Reviewers agreed at journal 22 Feb, 2025 Reviewers invited by journal 13 Feb, 2025 Editor assigned by journal 07 Feb, 2025 Editor invited by journal 07 Feb, 2025 Submission checks completed at journal 07 Feb, 2025 First submitted to journal 06 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5975519","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":423883178,"identity":"6af730fc-aede-4004-b7e4-61a37f650834","order_by":0,"name":"Evelyn Rivera-Toledo","email":"","orcid":"","institution":"Universidad Nacional Autónoma de México","correspondingAuthor":false,"prefix":"","firstName":"Evelyn","middleName":"","lastName":"Rivera-Toledo","suffix":""},{"id":423883179,"identity":"c092b400-37e5-44e7-97b3-8ccf1cafe28e","order_by":1,"name":"Fidencio Mejıa-Nepomuceno","email":"","orcid":"","institution":"Instituto Nacional de Enfermedades 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Kenneth Wickiser","email":"","orcid":"","institution":"Columbia University","correspondingAuthor":false,"prefix":"","firstName":"J.","middleName":"Kenneth","lastName":"Wickiser","suffix":""},{"id":423883195,"identity":"51c8e1b2-c194-4bbc-9784-8c7e3411ef64","order_by":17,"name":"Joel Armando Vazquez-Perez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACxgYogx9EJBSQokUSxEgwIMU6gwNgkgiVzO3NxyR+/LkjZ3x+deKHBwYM8vxiBwg4rOdYmmQPzzNjsxtvN0sAHWY4c3YCAS0zcowNeCQOJ267cXYDSEuCwW2CWvI/G/4xOFy/ecbZzT+I1JLD+Jgn4XCCAX/vNiJt6Tlm+FjmwDPDGTd4t1kkGEgQ9othe/ODg2/+3JHn7z+7+eaPCht5fmlCWhrA1AEGBgmwSgn8ykFAngGmhf8AYdWjYBSMglEwMgEAifxI8+/5qKcAAAAASUVORK5CYII=","orcid":"","institution":"Instituto Nacional de Enfermedades Respiratorias “Ismael Cosío Villegas”","correspondingAuthor":true,"prefix":"","firstName":"Joel","middleName":"Armando","lastName":"Vazquez-Perez","suffix":""}],"badges":[],"createdAt":"2025-02-06 17:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5975519/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5975519/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-13061-9","type":"published","date":"2025-07-28T16:13:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77890003,"identity":"a5f3eef8-49dc-43cf-a95f-a3cabe1c0298","added_by":"auto","created_at":"2025-03-06 13:45:19","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":143346,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood (ML) phylogenetic tree for RSV-A complete genome. ML tree from 1029 hRSV-A viruses registered in GISAID and the 81 Mexican sequences from 2022-2024 of this study (colored in red). Branches indicate the lineage classification and black star A.D lineage. Scale bar indicates substitutions per site.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5975519/v1/1c4785796329f876f827d687.png"},{"id":77888288,"identity":"2b1a80de-0373-4814-8783-2ec6dc9968da","added_by":"auto","created_at":"2025-03-06 13:29:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":80864,"visible":true,"origin":"","legend":"\u003cp\u003eMaximum likelihood (ML) phylogenetic tree for RSV-B complete genome. ML tree from 506 hRSV-B viruses registered in GISAID and the 29 Mexican sequences from 2022-2024 of this study (colored in red). Branches indicate the lineage classification and black star B.D lineage. Scale bar indicates substitutions per site.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5975519/v1/0d29f2a3b94b1a48910d7d24.png"},{"id":77888603,"identity":"0a04c439-849b-4f64-8840-512a0d5559e3","added_by":"auto","created_at":"2025-03-06 13:37:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1357937,"visible":true,"origin":"","legend":"\u003cp\u003eTotal substitutions in hRSV proteins and association with age groups. The heat maps represent substitutions identified in A) hRSV A, and B) hRSV B. Each row corresponds to individual hRSV A and hRSV B isolates (81 and 29, respectively), whereas columns represent substitutions in each of the eleven viral proteins. The numeric value in the last column indicates total mutations per virus isolate. Age ranges are indicated in the left side of the heat maps. C) Total substitutions per virus isolate with respect to age group. Median with interquartile range is shown. Differences between groups was analyzed by One-way ANOVA test; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5975519/v1/e669a2a64e7315dd4efc0147.png"},{"id":77888292,"identity":"d44dcb3e-34c3-4d36-8f68-fab1e83b8292","added_by":"auto","created_at":"2025-03-06 13:29:19","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":592453,"visible":true,"origin":"","legend":"\u003cp\u003eSubstitutions in hRSV A proteins. The bars represent amino acid positions with substitutions occurring at a frequency ³2.5% (at least 2/81 sequences).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5975519/v1/303368eab0910f33cf8d4075.png"},{"id":77888294,"identity":"e6618cc5-4899-44ee-beca-d727f52da220","added_by":"auto","created_at":"2025-03-06 13:29:19","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":347996,"visible":true,"origin":"","legend":"\u003cp\u003eSubstitutions in hRSV B proteins. The bars represent amino acid positions with substitutions occurring at frequencies ³6.9% (at least 2/29 sequences). Proteins NS2 and M2-1 each exhibited single mutation with frequency ³6.9%; both are represented in the same graph.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5975519/v1/dd8a5346b31058acbde5c62b.png"},{"id":77888296,"identity":"9fbe6f07-d6e8-4ccd-8769-04b864d7020d","added_by":"auto","created_at":"2025-03-06 13:29:19","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":886412,"visible":true,"origin":"","legend":"\u003cp\u003eSubstitutions in the most variable hRSV A proteins. The bars represent amino acid positions with substitution frequencies identified in A) the F protein, B) the G protein and C) the viral polymerase (L). Graph bar A) represents all mutations identified in the F protein of hRSV A, whereas B) and C) show only mutations with frequency values ³2.5%. The distribution of the main domains in each viral protein is also represented. Abbreviations: CT, cytoplasmic domain; TM, transmembrane domain; CD, conserved domain; HCD, highly conserved domain; HBD, heparin-binding domain; RdRp, RNA-dependent RNA polymerase; PRNTase, polyribonucleotidyl transferase or capping domain; MTase, methyltransferase domain; CTD, C-terminal domain.\u003c/p\u003e","description":"","filename":"Figure6ABC.png","url":"https://assets-eu.researchsquare.com/files/rs-5975519/v1/ee13de6959a7887c467ba1d9.png"},{"id":77888295,"identity":"9ab7ec1a-d40d-4edd-97af-0e6d410ec0bc","added_by":"auto","created_at":"2025-03-06 13:29:19","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":510572,"visible":true,"origin":"","legend":"\u003cp\u003eSubstitutions in hRSV B envelope proteins and viral polymerase. The bars represent amino acid positions with substitution frequencies identified in A) F protein, B) G protein, and C) viral polymerase (L). Graph bar A) represents all mutations identified in the F protein of hRSV B, whereas B) and C) display only mutations with frequencies ³6.9%.\u003c/p\u003e","description":"","filename":"Figure7ABC.png","url":"https://assets-eu.researchsquare.com/files/rs-5975519/v1/33f3a121041a9e7d6aa5863d.png"},{"id":88268299,"identity":"69d47bf9-2945-4867-8347-1fe4ee183238","added_by":"auto","created_at":"2025-08-04 16:50:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4913904,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5975519/v1/e7b0be30-60e4-4f53-8e7f-73c8c1cd9d8d.pdf"},{"id":77888291,"identity":"0934e761-66ab-4973-be83-addcb3a66866","added_by":"auto","created_at":"2025-03-06 13:29:19","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":72310,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5975519/v1/039ef28da0ebf5e4ae77eb91.xlsx"},{"id":77888287,"identity":"8a874525-8b31-4e19-bfc5-c0d0292d3973","added_by":"auto","created_at":"2025-03-06 13:29:19","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26780,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5975519/v1/93273cc72b36833a435ed5b2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Molecular characterization of human Respiratory Syncytial Virus in Mexico (season 2023–2024) through whole-genome sequencing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHuman respiratory syncytial virus (hRSV) is an enveloped virus from the \u003cem\u003ePneumoviridae\u003c/em\u003e family, which is a leading etiology of acute lower respiratory tract infection (ALRTI) in children under five years of age. Annually, hRSV produces approximately 33.0\u0026nbsp;million ALRTI, along with 3.6\u0026nbsp;million hospitalizations and 26,300 in-hospital deaths (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). In adults over 65 years of age hRSV-related hospitalizations are estimated from 356,000 to 466,000 in industrialized countries and up to 33,000 in-hospital deaths (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe hRSV genome consists of ten genes encoded in a single-stranded RNA of approximately 15,200 nucleotides (nt) in length that expresses eleven proteins: NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2 and L. The envelope glycoproteins G and F mediate viral attachment and entry to the host cells, respectively, and are major targets of the immune response.\u003c/p\u003e \u003cp\u003eThe G protein is expressed as a precursor of 32 kDa that is modified by N- and O-glycosylation becoming a mature protein of 80\u0026ndash;90 kDa (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). The heavily glycosylated sequence is arranged as an ectodomain comprising two highly variable mucin-like domains connected by a central conserved region (aa 163\u0026ndash;189) that includes a highly conserved sequence (aa 164\u0026ndash;176), and four cysteines maintained in all viral strains. Additionally, this central region contains a CX3C motif (aa 182\u0026ndash;186), which binds to both the fractalkine receptor to induce leukocyte chemotaxis and an heparan binding sequence (aa 184\u0026ndash;197) (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e)(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe F glycoprotein is synthesized as a precursor, F(0), which is cleaved by a furin-like protease into two subunits, F1 and F2, with the release of a 27-amino acid peptide. F1 and F2 remain linked by two disulfide bridges. F1 contains the fusion peptide that mediates virus entry through fusion of its envelope with the cellular membrane, following the interaction with the viral receptor (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Therefore, F exists in a metastable prefusion conformation and a highly stable postfusion structure. Six antigenic sites have been identified in F (denoted as \u0026oslash;\u0026ndash;V), of which sites \u0026oslash; and V are exclusively found in the prefusion state (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Interestingly, human neutralizing antibodies with high potency target the prefusion antigenic sites. As a result, the recently approved prophylactic monoclonal antibody Nirsevimab, as well as the vaccines Abrysvo and Arexvy, were designed to target the prefusion conformation of F (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTwo antigenic subgroups of hRSV, A and B, are recognized based on cross-neutralization assays and the sequence variation of the G gene, which exhibits the highest variability among the viral genes (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince its first isolation in 1956, this virus has undergone genetic diversification, leading to the identification of emergent genotypes and lineages. Particularly, it was identified in the period of June to August 1999 in Buenos Aires, Argentina, a 60-nt duplication (20 amino acid) in the G gene of hRSV B (Trento et al., 2003, DOI \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1099/vir.0.19357-0\u003c/span\u003e\u003cspan address=\"10.1099/vir.0.19357-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Also, in season 2010\u0026ndash;2011 in Ontario, Canada, it was observed a duplication of 72-nt (24 amino acid) in the G gene of a hRSV A isolate (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Currently, both variants are globally dominant and continue to experience genomic changes. Surveillance of hRSV whole-genome has gained relevance in the context of the newly approved prophylactic therapies to understand virus evolution and evaluate the potential emergence of immune escape variants.\u003c/p\u003e \u003cp\u003eStandardized criteria for the classification and genotyping of hRSV are essential in molecular epidemiology for monitoring the circulation of variants globally. Recently, Goya et al., proposed a phylogenetic classification using whole-hRSV genome sequences and a system based on amino acid markers to define distinct lineages (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHerein we describe the molecular characteristics of the hRSV circulating in Mexico City during the season 2023\u0026ndash;2024.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eSample Selection\u003c/p\u003e \u003cp\u003eAs part of a surveillance program at the Instituto Nacional de Enfermedades Respiratorias (INER), nasopharyngeal swabs of individuals presenting with Case of Influenza-Like Illness (ILI) and severe acute respiratory infection (SARI) are routinely collected. Patients with acute respiratory disease or chronic lung disease exacerbation requiring hospitalization, as well as ambulatory cases are sampled to be tested. One hundred and forty-three hospitalized patients, of which 100 were children and 43 adults, as well as 14 non-hospitalized adult patients were enrolled in this study. All patients were confirmed by the RT-qPCR diagnostic kit Respiratory Panel Filmarray (BioM\u0026eacute;rieux, Spain). hRSV subgroup identification (subtyping) and cycle threshold (Ct) values were determined by RT-qPCR, using in-house designed primers for detection of the nucleocapsid (N) gene (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eViral RNA extraction and whole genome amplification\u003c/p\u003e \u003cp\u003eWhole genome sequencing was performed in samples with a high viral load (Ct\u0026lt;25) to facilitate amplification and sequencing. Viral RNA was extracted from 200 \u0026micro;l of nasal swabs in viral transport media (VTM), using QIAamp Viral RNA mini kit (QIAGEN). The whole genome was amplified simultaneously and directly from clinical samples, using panel of primers modified and optimized for multiplex PCR (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). These primers were used to cover the complete hRSV genome (both A and B) by splitting into two pools of non-consecutive amplicons. An alternative method was used for hRSV-A positive samples from winter season 2022\u0026ndash;2023, alternative methodology was used (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). For the hRSV-B samples COI-5 and COI-7, which were identified as co-infections by initial PCR testing, the VirCapSeq-VERT (VCS) protocol was utilized to sequence the complete genomes of all vertebrate viruses (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). VCS data were analyzed with the Rapid Identification of Microbes (RIM), a custom viral metagenomics pipeline (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLibraries preparation and Sequencing\u003c/p\u003e \u003cp\u003eLibraries of hRSV complete genome were generated using the reagents of the Covid-Seq kit (Illumina, San Diego, CA, USA). Libraries were sequenced on a MiSeq sequencing platform using a 2 x 150-cycle to obtain paired-end reads (Illumina, San Diego, CA, USA). The DRAGEN COVIDSeq Targeted Microbial Pipeline on BaseSpace Sequence Hub performed the analysis, mapping, and consensus. For COI-5 and COI-7, libraries were prepared with Twist library preparation and fast hybridization reagents (Twist Bioscience, San Francisco, CA, USA), and sequenced on a NextSeq 2000 instrument using a 2 x 150-cycle to obtain single-end reads (Illumina, San Diego, CA, USA). Mapping and consensus were carried out with the RIM pipeline.\u003c/p\u003e \u003cp\u003ePhylogenetic and mutations analysis\u003c/p\u003e \u003cp\u003eTo perform phylogenetic analysis, we analyzed for hRSV-A 1,032 complete genomes and selected 877 sequences of different states in the USA from 2022 to 2024, 25 sequences of Panama from 2022\u0026ndash;2023 and 49 sequences of Canada, available on the GISAID platform. We analyze the 81 whole genome sequences of this study, including 19 sequences from winter season 2022\u0026ndash;2023. For hRSV-B, 506 complete genomes 436 sequences of different states in the USA from 2021 to 2024 and 41 from Nicaragua, available on the GISAID platform. We included the 29 whole genome sequences from this study. A maximum likelihood tree was constructed for the whole genome sequence using MEGA 10.0. The General Time-Reversible model was selected with five-parameter gamma-distributed rates and 1000 bootstrap replicates. Edition of the trees was made using FigTree (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). hRSV-A and hRSV-B clades were assigned by using the real-time phylogenetic analysis in Nextclade (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Non-synonymous substitutions were obtained using hRSV-A and hRSV-B reference sequence A/England/397/2017 accession number EPI_ISL_412866 and B/Australia/VIC-RCH056/2019 accession number EPI_ISL_1653999 respectively, in Nextclade (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eDuring the 2023–2024 season, a total of 100 pediatric patients (aged 1 month to 17 years) and 43 adult patients (mean age 55\u0026nbsp;±\u0026nbsp;18 years) with confirmed hRSV infection by RT-qPCR were hospitalized at the INER.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eComorbidities were identified in 68% of children, with the most common being malnutrition (18%), followed by recurrent wheezing (14%), gastroesophageal reflux diseases (12%), asthma (11%) and allergic rhinitis (11%) (Table 1).\u003c/p\u003e\n\u003cp\u003eSubgroup identification was performed in 88/100 pediatric samples, of which 68% were positive for hRSV A and 20% positive for hRSV B. Additionally, 13% of pediatric samples tested positive for hRSV by Viral Respiratory Panel (Filmarray) but could not be further subtyped by the specific RT-qPCR assay.\u003c/p\u003e\n\u003cp\u003eIn hospitalized adults, the most common comorbidities were chronic lung disease, asthma, COPD, IFD, obesity and type 2 diabetes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ehRSV phylogeny\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWhole hRSV genome sequencing was achieved of 70 respiratory samples from pediatrics and 40 from adults (Table 2), of which, 81 correspond to hRSV-A and 29 to hRSV-B. These sequences included; 14 of non-hospitalized adult patients from winter season 2023-2024 and 19 from winter season 2022-2023 (GISAID accession number\u0026nbsp;EPI_ISL_19500979- EPI_ISL_19501009 and EPI_ISL_19504707-EPI_ISL_19504788). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis indicated that all hRSV-A isolates belonged to the A.D lineage (which carry the duplication of 72 nt in the G gene) and ten sublineages (Fig 1). The sublineages A.D.1.5, A.D.1.8, A.D.3, and A.D.5.2 were prevalent (66/81 isolates, Table 3), mainly in children under 5 years old. The analysis using complete genome showed that Mexican sequences collected in winter season 2023-2024 formed 3 different groups: A.D.1 and sub-lineages; A.D.3 and sub-lineages, and A.D.5 and sub-linages (Fig 1). Remarkably, two sequences of 2023 belong to clade A.D. which is the ancestor of the newest sub-lineages. Each group clustered with sequences from USA from 2022-2024, 2023-2024 and 2022-2024 respectively (Fig 1). The hRSV-B sequences were less diverse (Fig 2), with all belonging to the B.D lineage (characterized by the 60 nt duplication in the G gene) whereas the dominant sub-lineage was B.D.E.1 (28/29 isolates). Mexican sequences (B.D.E.1 lineage) clustered with 2022-2024 sequences from USA (Fig 2). Lineage-defining amino acids in Mexican sequences were present mainly in G, L and F genes and are summarized in Table 4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAmino acid substitutions in hRSV proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total number of amino acid substitutions per hRSV isolate is shown in the heat maps in Figure 3. Among the 81 hRSV-A sequenced samples, it was found a median value of 43 amino acid substitutions per viral genome (range 23–88, Fig. 3A), while for the 29 hRSV B positive samples the median value was of 31 substitutions per viral genome (range 0–60, Fig. 3B). Analysis between age groups showed a significantly increased number of substitutions in hRSV-A isolates from younger individuals, 0–5 years old, with respect to the 22–49 years old group. In contrast, the total number of substitutions in hRSV-B was not significantly different between either age group (Fig. 3C).\u003c/p\u003e\n\u003cp\u003eThe highest variability was observed in the G glycoprotein (Fig. 3A and B) from both hRSV subgroups. Table 5 summarizes the total number of all the diverse substitutions identified in the eleven encoded proteins from virus isolates. Although the highest number of substitutions was identified in the L protein, the substitution density, which is related to the length of each protein, was higher in the protein G, followed by protein M2-2 in hRSV-A and SH in hRSV-B. Interestingly, hRSV-A showed more variation than hRSV-B, as the calculated hRSV-A/hRSV-B substitution ratio was \u0026gt;1 for each protein, excepting for the M protein with ratio of 0.8.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral amino acid substitutions were exclusive to individual genome and all of them are reported in Additional_file_1. Figures 4 and 5 show amino acid substitutions identified in at least two viral genomes, particularly in proteins NS2, N, P, M, SH, M2-1 and M2-2. The lowest number and frequency of mutations was identified in the protein NS1 from hRSV-A and the phosphoprotein P from hRSV-B (Additional_file_1). In contrast, mutations with frequencies \u0026gt;40% were detected in proteins N, P, M, M2-1, and M2-2 of hRSV-A, as well as in proteins N and M2-2 of hRSV-B (Fig. 4 and 5).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubstitutions in the envelope glycoproteins that mediate viral entry and the polymerase were diverse and many of those displayed frequencies higher than 50% (Figure 6), except in the F protein of hRSV-A. More frequent amino acid substitutions in the glycoprotein F of hRSV-A were localized in the signal region (T12I, T13A, L15F), F2 (I59V, A103T), p27 (L119F, T122A, N124I, V127I), as well as in the ectodomain (K470R, A518V) and the cytoplasmic region (R553K) of F1 (Fig. 6A). Low frequency substitutions were identified in the fusion peptide (S146P, I148S, V154I), the antigenic site ø (I206A), the antigenic site II, specifically in the binding site of Palivizumab (L258I, N262K and S276N) and the transmembrane region (V536K). The F protein of hRSV-B showed less variability than that of hRSV-A, although some substitutions were detected with frequencies close to 100%. Of those, three were localized to the ectodomain of F1 (S190N, S211N, S389P) and particularly, S211N was localized in the antigenic site ø (Fig. 7A). Other two low frequency substitutions in site ø were M206I and R209Q.\u003c/p\u003e\n\u003cp\u003eIn the glycoprotein G, most substitutions were localized, as expected, to the mucin-like regions 1 and 2. Interestingly, G from hRSV-A showed three substitutions in the central conserved region (F168Y, S177G, N178G) of which, F168Y was within the highly conserved domain (HCD, Fig. 6B). In hRSV-B the substitution T198I with frequency of 10% was also localized in the central conserved region (Fig. 7B).\u003c/p\u003e\n\u003cp\u003eFinally, the viral polymerase displayed most substitutions in the RdRp domain of both hRSV-A and hRSV-B (102 and 36 substitutions, respectively), whereas the less variable region was the catalytic domain with MTase activity (Fig 6C and 7C; Supplementary material). Positions R1339 and G1855 have been described relevant for capping and MTase activity, respectively (18), and we identified substitutions R1339P and G1855R in our hRSV-A isolates (Additional_file_1).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eHospitalizations due to acute hRSV lower respiratory tract infections during the 2023–2024 season showed that infants under five years of age were the primary affected group.\u003c/p\u003e\n\u003cp\u003ehRSV-A exhibited a higher prevalence compared to hRSV B in both pediatric and adult patients. Particularly, infections by hRSV-A outnumbered those by hRSV-B by a factor of 3.4 in pediatric patients and approximately 2.1 in adults (Tables 1 and 2). Previous studies have documented that hRSV-A replicates faster and at higher titers (up to 2 log\u003csub\u003e10\u003c/sub\u003e PFU/ml) than hRSV B in airway human epithelial cell cultures (19) (20). However, similar viral loads have been found in respiratory samples from infants infected with either subgroup A or B, as measured by RT-PCR or viral titration (21) (22), suggesting that factors besides replication rate contribute to the higher prevalence of hRSV-A.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOur phylogenetic analysis showed that the prevalent lineages of hRSV-A were A.D.1.5, A.D.1.8, A.D.3, and A.D.5.2, while for hRSV-B, the predominant lineage was B.D.E.1. Studies conducted in three different countries reported the prevalence of the following clades after season 2021–2022: A.D.3, A.D.5, A.D.5.2 and B.D.E.1 in USA (Minnesota); A.D.3, A.D.5.2, B.D.4.1.1 and B.D.E.1 in China (Beijing); and A.D.1, A.D.3.1, A.D.5.2 and B.D.E.1 in Italy (Sicily) (23) (24) (25). Accordingly, A.D.3, A.D.5.2 and B.D.E.1 may have global distribution (25). Notably, the study from Beijing, China reported that B.D.E.1 became prevalent since November 2023 and affected a significantly higher proportion of patients ≥60 years (3-fold increase) than did its parent lineage B.D.4.1. However, B.D.E.1 was mostly associated with development of upper respiratory tract infections and non-severe pneumonia, conversely to the severe diseases caused by the previously dominant lineages A.D, A.D.3 and B.D.4.1 (25).\u003c/p\u003e\n\u003cp\u003eAmino acid substitutions per viral genome and the substitution ratio (hRSV-A/hRSV-B) for each protein indicated higher variability in hRSV-A compared to hRSV-B. Total substitutions were significantly higher for hRSV-A infecting the 0–5-year-old age group than in the 22–49-year-old group. This difference may be at least partially explained by the prevailing circulation of the hRSV-A in children, a host population with a naïve or minimally experienced immune system. This condition facilitates higher viral replication, which in turn can lead to an increased mutation rate (26) (27).\u003c/p\u003e\n\u003cp\u003eAs expected, we observed a high diversity of substitutions in proteins G, F and L. However, all substitutions in the F protein showed frequencies \u0026lt;40%, whereas many substitutions in proteins G and L displayed frequencies of up to100%.\u003c/p\u003e\n\u003cp\u003eThe F protein contains six potential N-glycosylation sites located at positions N27, N70, N116, N120, N126 and N500. N116 and N126 are within the p27 fragment, which is cleavaged by furin-like cellular proteases (28), while N120 is not conserved among different hRSV isolates (29). Both, the double mutant N27Q/N70Q and the individual N500Q impair membrane fusion (29). We did not identify substitutions in the N-glycosylation sites of our viral isolates. However, we observed the substitution T122A (with a frequency of 28.2%), located in the consensus N-glycosylation sequence N-X-T/S. It has been suggested that T122A might reduce the glycosylation at N120, although this likely has no significant impact on fusion activity (29).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, we also detected substitutions in the F protein located within the antigenic sites ø and II. Particularly, the low frequency substitutions L258I and N262K were found in the Palivizumab binding site (residues 258 to 275) of hRSV-A, along with the adjacent substitution S276N. Zhu et al., reported 46 polymorphic sites in the extracellular region of the F protein of clinical isolates from children without prophylactic treatment, (including the variation at position 276 (30). Using microneutralization assays, they determined that only the substitutions N262D and S275F conferred resistance to Palivizumab. These substitutions were identified in 2 of 145 hRSV A isolates by the authors and were considered as natural polymorphisms. In our study, the substitution N262K was identified only in 1 of 81 hRSV-A isolates.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePalivizumab resistance has been observed in 5–10% of immunocompromised infants with long-term infections and under treatment with Palivizumab. In such cases, substitutions N262D, K272E and S276N have been reported, although only the first two were associated with Palivizumab resistance (31) (30).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe recently approved prophylactic monoclonal antibody Nirsevimab targets a prefusion discontinuous neutralizing epitope within site Ø, spanning residues 62 to 69 and 196 to 212 (32). Although most residues within the binding site are conserved at a frequency of \u0026gt;99%, a partial reduction in susceptibility to Nirsevimab has been associated with specific substitutions. These include N208D/S, K65Q/T and the dual substitution N67I/N208Y in hRSV-A, or dual substitutions K68N/N201S and K65Q/S211N in hRSV-B (33) (34).\u003c/p\u003e\n\u003cp\u003eIn our study, we identified the substitution I206A (3.8%) in hRSV-A, and the substitutions M206I (3.4%), R209Q (6.9%), and S211N (96.6%) in hRSV-B. These substitutions within the Nirsevimab binding site have been documented as natural polymorphisms, and through microneutralization assays it has been determined that changes in the position 206 of the hRSV A-F protein do not modify the neutralization activity of Nirsevimab, while single mutations in positions 206 and 209 only partially reduce neutralization activity against hRSV-B isolates (34).\u003c/p\u003e\n\u003cp\u003eOther substitutions in F, localized in the signal peptide, p27, transmembrane and cytoplasmic domains showed frequencies \u0026gt;13%. Coincident with our observations it has been previously reported that domains with the greatest number of non-synonymous changes and amino acid positions with higher entropy values are within the signal peptide, p27, heptad repeat domain 2, antigenic site ø, and the transmembrane domain (35).\u003c/p\u003e\n\u003cp\u003eGiven the high variability of the antigenic site ø (mainly in hRSV-B), it is of relevance a continued surveillance of emerging variants besides the recently approved anti-hRSV vaccines and the monoclonal antibody Nirsevimab (36).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs predicted, the G glycoprotein showed high variability, primarily in the second mucin-like region. However, we also identified the substitutions F168Y, S177G and N178G in the central conserved domain (CCD) of hRSV-A and substitution T198I in hRSV-B. \u0026nbsp;Attachment of hRSV to primary respiratory epithelial cells occurs by binding of the G protein to CX3CR1 and infection is attenuated in viruses lacking the G protein or with mutations in the CX3C motif (37). Although the highly glycosylated domains of the G protein are poor immunogens, antibodies against epitopes within the CCD have been detected. These antibodies can induce antibody-dependent cellular cytotoxicity or block the CX3C–CX3CR1 interaction (38) (39). Furthermore, substitution as 177Q and 177R in the CCD enhance G protein immunogenicity and induce IgG antibodies that inhibit the CX3C–CX3CR1 interaction, thereby reducing pulmonary cell infiltration and lung damage in mice (40). In our study, we identified the substitution S177G at a low frequency (3.7%) in hRSV-A isolates. It would be of interest to evaluate if the serum from individuals infected with this variant can reduce hRSV infection of primary airway epithelial cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOn the other hand, Li et al., previously reported that substitutions T113I, V131D, N178 G, H258Q and H266 L in the G protein are associated with decreased disease severity in hospitalized infants (41). We found the same five substitutions in hRSV-A isolates, with frequencies of 13.6% to 17.3%. Further studies are necessary to assess the impact of these substitutions on viral infectivity. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRegarding the L protein or RNA-dependent RNA polymerase, we identified 308 and 85 different non-synonymous substitutions in hRSV-A and hRSV-B isolates, respectively. This enzyme is multifunctional, as it not only participates in the transcription and replication of the viral genome but also exhibits polyribonucleotidyltransferase (PRNTase) activity to add the cap structure, as well as methylase activity to methylate the cap (42). Certain mutations in the L protein have been identified in the context of studies with antiviral drugs that inhibit its enzymatic activity (43) (44) (45). In our analysis, we did not identify substitutions associated with antiviral drug resistance. However, we observed substitutions R1339P and G1855R within the catalytic pocket of the capping domain and the SAM/SAH GxGxGx binding motif of the MTase domain, respectively (18). Both substitutions were present at a frequency of 1.2%. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSubstitutions in hRSV proteins other than F, G and L have been less extensively studied. Nevertheless, the growing availability of complete hRSV genome sequences will facilitate the surveillance of specific substitutions and their frequencies, thereby contributing to a better understanding of viral evolution and the effectiveness of prevention and treatment strategies.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eWe thank Eduardo Márquez García from the Unidad de\u0026nbsp;Biología\u003c/p\u003e\n\u003cp\u003eMolecular,\u0026nbsp;INER for technical assistance in Illumina sequencing. Also, to Carlos Santiago-Olivares and Ana Flisser for technical assistance and provide equipment and materials respectively. We also thank all physicians in the ICU for\u0026nbsp;assistance with patient management.\u003c/p\u003e\n\u003cp\u003eAuthors´contributions\u003c/p\u003e\n\u003cp\u003eERT, JRP, JKW and JAVP conceived and designed the project. VHAT, MCL, FBPO, and\u003c/p\u003e\n\u003cp\u003eMASL collected the clinical data and constructed the database. ERT, FMN, AVJ, EMR, JPC\u003c/p\u003e\n\u003cp\u003eand JVP performed the experimental laboratory procedures. ERT, XJL and EMR\u003c/p\u003e\n\u003cp\u003eperformed the bioinformatics and statistical analyses. EBV, FBPO, GB, JDCA, EGC and CGG performed the interpretation of clinical data. ERT, JKW, VHAT, and\u003c/p\u003e\n\u003cp\u003eJAVP wrote the manuscript. JAVP supervised the project and led the team. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003eAvailability of data and material\u003c/p\u003e\n\u003cp\u003eThe genomic information generated during the current study is available in GISAID database. Sequences of RSV-A and RSV-B from Mexico were deposited in GISAID under accession number\u0026nbsp;EPI_ISL_19500979- EPI_ISL_19501009 and EPI_ISL_19504707-EPI_ISL_19504788.\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests. The sponsors had\u0026nbsp;no role in the design, execution, interpretation, or writing of the study.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eInstitutional Review Board Statement: This study was reviewed and approved by the Science,\u0026nbsp;Biosecurity, and Bioethics Committee of the Instituto Nacional de\u0026nbsp;Enfermedades Respiratorias\u0026nbsp;(protocol number B22-23). The research carried out on humans and/or human data is in compliance with the Helsinki Declaration. Written informed consent was obtained from the patients and/or from their relatives or\u0026nbsp;authorized legal guardians prior to the publication of this paper.\u003c/p\u003e\n\u003cp\u003eFunding\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis study was financially supported by the Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT), Mexico, project CBF2023-2024-2356. This work was financially supported also by Direccion General de Politicas de Investigacion en Salud (DGPIS), Grant “FPIS2024-INER-4886” to J.A.V.-P. The use of VirCapSeq-VERT and related work at Columbia University’s Global Alliance for Preventing Pandemics (GAPP) were supported by the Skoll Foundation (J.K.W., J.P.C., and X.J.L.).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi, Y. et al. 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Interaction Between the Matrix Protein and the Polymerase Complex of Respiratory Syncytial Virus. \u003cem\u003eViruses\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e (12), 1881 (2024).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 5 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Human respiratory syncytial virus, whole-genome sequencing, phylogeny, lineages","lastPublishedDoi":"10.21203/rs.3.rs-5975519/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5975519/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eHuman respiratory syncytial virus (hRSV) is a one of major cause of severe acute respiratory infection (SARI) mainly in young children and the elderly. Genomic surveillance of hRSV is currently of interest for understanding of viral evolution and the monitoring of genetic variations that may affect transmissibility and pathogenicity. Herein, we sequenced complete genomes of hRSV-A and B from season 2023\u0026ndash;2024, isolated from pediatric and adult patients with SARI.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eOne hundred pediatrics and 43 adult hospitalized patients, as well as 14 non-hospitalized adult patients positive to hRSV were enrolled. Libraries of hRSV complete genome were generated and sequenced on a MiSeq platform. Phylogenetic analysis and maximum likelihood trees were constructed with the 81 hRSV A and 29 hRSV B sequences obtained in our study. Additionally, we analyzed the list of non-synonymous substitutions and their frequencies for each of the eleven viral proteins.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003ehRSV A was prevalent (68%) and children under five years old was the principal group affected. The hRSV A isolates belonged to the A.D lineage and sub-lineages A.D.1.5, A.D.1.8, A.D.3, and A.D.5.2 were prevalent. The hRSV B subgroup was less diverse since the dominant sub-lineage was B.D.E.1. Amino acid substitutions per viral isolate for each of the eleven viral proteins indicated higher variability in hRSV A compared to hRSV B. As expected, we observed a high diversity of substitutions in proteins G, F and L.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSeveral lineages and high rate of mutation mainly in RSV-A were found during winter season 2023\u0026ndash;2024 in Mexico. The increasing availability of complete hRSV genome sequences will facilitate the surveillance of specific substitutions, thereby contributing to a better understanding of viral evolution and the effectiveness of prophylactic strategies.\u003c/p\u003e","manuscriptTitle":"Molecular characterization of human Respiratory Syncytial Virus in Mexico (season 2023–2024) through whole-genome sequencing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-06 13:29:14","doi":"10.21203/rs.3.rs-5975519/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-26T19:40:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-23T21:35:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-14T10:57:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-06T13:14:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58215375887148194682097978714153374814","date":"2025-03-04T09:47:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230547144793308012550928554896591174228","date":"2025-02-25T13:26:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289322742139006990528706823769640810972","date":"2025-02-22T23:22:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-13T09:17:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-07T13:10:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-02-07T09:51:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-07T05:08:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-02-06T17:19:03+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"478e53a3-9d35-40e2-b89f-328f8e6d300c","owner":[],"postedDate":"March 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":45169406,"name":"Biological sciences/Microbiology/Virology"},{"id":45169407,"name":"Biological sciences/Immunology/Infectious diseases/Viral infection"}],"tags":[],"updatedAt":"2025-08-04T16:42:53+00:00","versionOfRecord":{"articleIdentity":"rs-5975519","link":"https://doi.org/10.1038/s41598-025-13061-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-28 16:13:18","publishedOnDateReadable":"July 28th, 2025"},"versionCreatedAt":"2025-03-06 13:29:14","video":"","vorDoi":"10.1038/s41598-025-13061-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-13061-9","workflowStages":[]},"version":"v1","identity":"rs-5975519","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5975519","identity":"rs-5975519","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}