Genomic Sequencing Surveillance and Antigenic Site Mutations of Respiratory Syncytial Virus in Arizona, USA

RSV, Arizona, Genomic Surveillance, Antigenic sites 16 17 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2023. ; https://doi.org/10.1101/2023.06.16.23291299doi: medRxiv preprint NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice. 2

18 We conducted Respiratory Syncytial Virus (RSV) genomic sequencing surveillance of 19 100 RSV-A and 27 RSV-B specimens collected between November 2022 and April 2023 in 20 Arizona, USA. We identified mutations in the pre-fusion F protein antigenic sites in both RSV-A 21 and RSV-B. Continued genomic surveillance will be important to ensure RSV vaccine 22 effectiveness. 23 24 Research 25 Respiratory syncytial virus (RSV) is an RNA virus of the Paramyxoviridae family which 26 causes acute respiratory infections primarily in children, adults with severe lung disease, and the 27 elderly (1). The United States experienced an early surge in cases of RSV during the 2022-2023 28 respiratory pathogen season (2). The surge in RSV infections coincided with high circulating 29 levels of influenza and SARS-CoV-2 viral infections, particularly in children (2). In Arizona, 30 USA, laboratory-confirmed RSV cases increased from September 2022 through March 2023 31 with cases peaking in mid-November (Figure 1A). 32 The 2 major subtypes of RSV, RSV-A and RSV-B, have distinct antigenic differences in 33 the P, N, F and G proteins (1). Each subtype is classified into genotypes based on sequence 34 variability in the G protein (3). Based on sequencing data, the global distribution of RSV 35 genotypes in 2022-2023 was split between the GA2.3.5 genotype of RSV-A and the GB5.0.5a 36 genotype of RSV-B (Figure 1B). However, currently approved RSV vaccines are based on 37 prefusion conformation of the F protein. Therefore, RSV genotypes do not reflect circulating 38 RSV strains that harbor F protein mutations which can affect vaccine responses. Thus, genomic 39 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2023. ; https://doi.org/10.1101/2023.06.16.23291299doi: medRxiv preprint 3 sequencing surveillance is needed to better understand the evolution of the virus and its potential 40 impact on vaccine efficacy. 41 We performed genomic sequencing surveillance of RSV circulating in Arizona, USA, 42 during the 2022-2023 season using remnant RSV-positive nasopharyngeal swabs (N=127) 43 collected as part of standard-of-care respiratory pathogen testing at Valleywise Health (Figure 44 1A), which serves the population of Maricopa County. Next-generation sequencing (Illumina, 2 45 x 150 paired end) was performed using a hybrid-capture method targeted for the RSV genome 46 (Illumina Respiratory Virus Oligo Panel v2). Sequencing reads were quality filtered, adapter 47 trimmed (Trim Galore version 0.6.10), mapped to RSV-A and RSV-B reference sequences 48 (GISAID EPI_ISL_412866 and EPI_ISL_165399) (Burrows-Wheeler Aligner version 0.7.17-49 r1188), and consensus sequences generated (SAMtools version 1.17). We assembled the 50 complete genome sequences of 92 RSV-A (GA2.3.5 genotype) and 24 RSV-B (GB5.0.5a 51 genotype), and partial genome sequences of 8 RSV-A (GA2.3.5) and 3 RSV-B (GB5.0.5a) 52 (GenBank Accessions: OR143134 - OR143250; GISAID Accessions: EPI_ISL_17808760 - 53 EPI_ISL_17808814). To determine RSV viral load, we performed qRT-PCR assays (HRSV-pan) 54 that recognizes both subtypes RSV-A and RSV-B (4). The mean RSV CT value was 29.83 with a 55 standard deviation of 7.44. We found that specimens with viral load CT’s ≤ 33 yielded 99-100% 56 genome coverage (Figure 1C). Whole genome phylogenetic analysis (Nextclade version 2.14.1) 57 showed that the Arizona RSV-A sequences were polyphyletic in the GA2.3.5 clade indicating at 58 least 3 independent introductions of RSV-A into Arizona (Figure 1D). The Arizona RSV-B 59 sequences formed a monophyletic clade in GB5.0.5a indicating a single introduction that seeded 60 local transmission within the state (Figure 1D). Our findings are consistent with investigations 61 of RSV in Massachusetts (5) and Washington (6) that infer that the atypical increase in cases 62 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2023. ; https://doi.org/10.1101/2023.06.16.23291299doi: medRxiv preprint 4 during the 2022-2023 season was the result of multiple introductions of extant lineages and not 63 attributable to a divergent RSV lineage with increased virulence or transmissibility. 64 To date, two RSV vaccines (Arexvy and Abrysvo) have been FDA approved for 65 individuals 60 years and older in the US. Both vaccines are based on the RSV prefusion F 66 protein with Arexvy being monovalent and Abrysvo bivalent (7, 8). The majority of host 67 antibodies target six antigenic sites of the F protein, characterized as sites Ø-V (9). We examined 68 the F gene sequences of the Arizona RSV genomes and identified 7 non-synonymous 69 substitutions in antigenic sites I, II, IV, and V of RSV-A (Table 1). Similarly, we identified 5 70 non-synonymous substitutions in the antigenic sites Ø, I, II, and V of RSV-B. All mutations in 71 RSV-A genomes were found in low frequency, with no mutations observed in greater than 9% of 72 samples. Conversely, in the RSV-B genomes, most presented with high frequency mutations and 73 only one rare SNP was found. These trends in mutation frequencies were comparable to the 74 frequency in global RSV genome sequences. Finally, we mapped the mutations onto the pre-75 fusion F protein crystal structure (PDB: 7KQD). The structural model overlay revealed that the 76 mutations are exposed on the F protein surface suggesting that they may interfere with antibody 77 binding (Figure 1E). 78 Although RSV remains a significant clinical burden, the recently approved RSV vaccines 79 reduce the risk of lower respiratory tract illness. By tracking the evolution of RSV, we can 80 improve the design of vaccine formulations to improve vaccine effectiveness. A limitation of this 81 study is that we do not yet understand the functional consequences of the mutations identified in 82 the F protein antigenic sites. Nonetheless, our surveillance revealed sequence diversity in the F 83 protein antigenic sites that is not reflected in current genotyping schema, which is based on G 84 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2023. ; https://doi.org/10.1101/2023.06.16.23291299doi: medRxiv preprint 5 protein sequences. Overall, this study demonstrates the importance of genomic sequencing 85 surveillance for RSV and other pathogens of interest to clinical and public health. 86 87

88 We gratefully acknowledge Sarah Namdarian for assistance with the collection of the clinical 89 specimens, Alexis Thomas, Gabrielle Hernandez Barrera, Michelle Tan for assisting in specimen 90 processing, and Regan Sullins for assisting with library construction. We thank the authors from 91 originating laboratories responsible for obtaining the specimens and the submitting laboratories 92 where genetic sequence data were generated and shared via the GISAID initiative and NCBI 93 GenBank. 94 This study was approved by the Arizona State University Institutional Review Board 95 (STUDY00011967) and was supported in part by Arizona State University, and the Centers for 96 Disease Control and Prevention (CDC BAA 75D30121C11084). 97 Data availability 98 RSV genome sequences have been deposited into the GenBank database under accession 99 numbers OR143134 - OR143250; and the GISAID EpiRSV database under accession numbers 100 EPI_ISL_17808760 - EPI_ISL_17808814. 101 Competing interests 102 The authors declare no competing interests. 103 Contributions 104 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2023. ; https://doi.org/10.1101/2023.06.16.23291299doi: medRxiv preprint 6 Conceptualization: E.S.L.; Formal analysis: L.A.H., S.C.H., M.F.S., V.R.L.; Investigation: 105 L.A.H., S.C.H., M.F.S., V.R.L., E.S.L.; Resources: V.M., L.N., M.M., R.S., M.W.; Data 106 curation: L.A.H., S.C.H., M.F.S., V.R.L.; Writing-original draft: L.A.H., S.C.H., M.F.S., E.S.L.; 107 Writing-review and editing: L.A.H., S.C.H., M.F.S., L.N., E.S.L.; Supervision: E.S.L.; Funding 108 acquisition: E.S.L. All authors reviewed and approved the final manuscript. 109 110

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Kampmann B, Madhi SA, Munjal I, Simões EAF, Pahud BA, Llapur C, et al. Bivalent 133 Prefusion F Vaccine in Pregnancy to Prevent RSV Illness in Infants. New England Journal of 134 Medicine. 2023;388(16):1451-64. 135 9. Gilman MS, Castellanos CA, Chen M, Ngwuta JO, Goodwin E, Moin SM, et al. Rapid 136 profiling of RSV antibody repertoires from the memory B cells of naturally infected adult 137 donors. Sci Immunol. 2016 Dec 16;1(6). 138 139 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2023. ; https://doi.org/10.1101/2023.06.16.23291299doi: medRxiv preprint 8 140 Figure 1. Genomic sequencing analysis of RSV in Arizona, USA, 2022-2023. (A) The 5-week 141 moving average of PCR confirmed RSV detections in Arizona as reported by the National 142 Respiratory and Enteric Virus Surveillance System (NREVSS) is shown alongside RSV 143 sequence counts by genotype obtained for specimens utilized in this study. (B) The relative 144 abundance of RSV-A and RSV-B genotypes is shown for all RSV genomes deposited in GISAID 145 with collection dates between 1/1/2022 – 5/1/2023 (RSV-A n = 1,047; RSV-B n = 941) 146 including genotypes obtained for specimens utilized in this study (RSV-A n = 100; RSV-B n = 147 27). (C) RSV RT-PCR Ct values and genome coverage for RSV-A (red) and RSV-B (blue) 148 samples. (D) Phylogeny of the RSV-A GA2.3.5 (top) and RSV-B GB5.0.5a clades (bottom). 149 Nodes and colored branches highlight Arizona sequences. (E) Structure of RSV prefusion F 150 protein with surface exposed RSV-A (left) and RSV-B (right) SNPs found in Arizona sequences 151 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2023. ; https://doi.org/10.1101/2023.06.16.23291299doi: medRxiv preprint 9 labeled and highlighted in bold red. One protomer of each trimer model is colored by antigenic 152 site, two additional protomers shown in white. 153 154 Table 1: Non-synonymous amino acid substitutions in RSV-A and RSV-B F protein antigenic 155 sites found in Arizona genome sequences compared to global genome sequences. 156 RSV-A Mutation Antigenic site Arizona frequency (n=92) Global frequency (n=952) I57V Site V 2 (2%) 20 (2%) I59V Site V 1 (1%) 1 (0%) S276N Site II 4 (4%) 162 (17%) V379A Site I 8 (9%) 2 (0%) L381I Site I 1 (1%) 0 (0%) P389S Site I 2 (2%) 4 (0%) K470R Site IV 1 (1%) 0 (0%) RSV-B Mutation Antigenic site Arizona frequency (n=24) Global frequency (n=894) R42K Site I 18 (75%) 95 (11%) S190N Site V 24 (100%) 555 (77%) S211N Site Ø 24 (100%) 554 (76%) E378D Site III 2 (8%) 0 (0%) S389P Site I 24 (100%) 566 (78%) 157 . CC-BY-NC-ND 4.0 International licenseIt is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted June 20, 2023. ; https://doi.org/10.1101/2023.06.16.23291299doi: medRxiv preprint