Recurrent intrahost <em>G</em>-gene haplotype structures suggest potential co-transmission in RSV-A

Jingqi Yang; Yuhua Liao; Luyao Qin; Haoyang Wang; Yun Ma; Ye Tong; Fei Deng; Liguo Zhu; Hang-Yu Zhou; Aiping Wu

doi:10.1016/j.virs.2026.10.003

October 2026

. doi: 10.1016/j.virs.2026.10.003

Citation: Jingqi Yang, Yuhua Liao, Luyao Qin, Haoyang Wang, Yun Ma, Ye Tong, Fei Deng, Liguo Zhu, Hang-Yu Zhou, Aiping Wu. Recurrent intrahost G-gene haplotype structures suggest potential co-transmission in RSV-A .VIROLOGICA SINICA, 2026, 41(5) : 1054-1065. http://dx.doi.org/10.1016/j.virs.2026.10.003

A型呼吸道合胞病毒宿主内反复出现的G基因单倍型结构提示潜在共同传播

杨镜琪 ^a ,
廖宇华 ^a ,
秦璐瑶 ^a ,
王皓阳 ^a ,
马运 ^a ,
童叶 ^b ,
邓斐 ^b ,
朱立国 ^b,, ,
周航宇 ^a,, ,
吴爱平 ^a,,

1中国医学科学院系统医学研究院/苏州系统医学研究所, 重大疾病共性机制研究全国重点实验室, 中国, 江苏, 苏州 215123;
2 江苏省疾病预防控制中心急性传染病防制所, 江苏南京 210009

通讯作者： 朱立国, zhuliguo2002@163.com; 周航宇, zhy@ism.cams.cn; 吴爱平, wap@ism.cams.cn
收稿日期： 2026-07-15
录用日期： 2026-10-03

摘要

呼吸道合胞病毒（RSV）是全球婴幼儿住院的主要病因之一。其分子流行病学研究主要依赖G基因一致序列，而这类序列仅能反映每次感染中的优势病毒。深度测序揭示了宿主体内更为丰富的变异；明确这些变异之间的连锁关系，可为传播研究提供更精细的视角。本研究基于两个相互独立的RSV-A深度测序数据集（澳大利亚，n = 37；美国，n = 72）重建G基因单倍型。我们利用读段水平的连锁信息对变异进行定相，并将各单倍型定位于全球系统发育树上。超过90%的样本携带多个单倍型，且这些单倍型常位于不同的系统发育分支。每个数据集中均有一对跨分支组合占主导地位：澳大利亚数据集中C4+C6见于37份样本中的23份（62%），美国数据集中C18+C21见于72份样本中的26份（36%）。两组组合的重复出现频率均显著高于按月份和地区匹配的置换检验所预测的随机共现水平（P均为5.0 × 10^-⁵）。突变概率计算提示，宿主内反复发生的新生突变不太可能完全解释上述模式，尤其是C4+C6所涉及的5个连锁位点（概率≤5.36 × 10^-¹⁵）。反复共同引入既有单倍型可提供更简洁的解释，未来可通过传播链采样直接检验这一假设。所有分支定义位点均位于G基因第二高变区（G-HVR2）。在本研究检索的全球G基因一致序列集合中，可检测到优势分支状态，但按本研究标准未检测到少数分支的位点组合。因此，单倍型分辨率分析能够将低于一致序列水平的多样性转化为具有连锁关系且可解释的流行病学信号。
- A型呼吸道合胞病毒
- , G基因
- , 宿主内多样性
- , 宿主内进化
- , 单倍型重建
- , 变异连锁

Recurrent intrahost G-gene haplotype structures suggest potential co-transmission in RSV-A

Jingqi Yang ^a ,
Yuhua Liao ^a ,
Luyao Qin ^a ,
Haoyang Wang ^a ,
Yun Ma ^a ,
Ye Tong ^b ,
Fei Deng ^b ,
Liguo Zhu ^b,, ,
Hang-Yu Zhou ^a,, ,
Aiping Wu ^a,,

a. State Key Laboratory of Common Mechanism Research for Major Diseases, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, 215123, China;
b. Department of Acute Infectious Disease Control and Prevention, Jiangsu Provincial Center for Disease Control and Prevention, Nanjing, 210009, China

Corresponding author: Liguo Zhu, zhuliguo2002@163.com Hang-Yu Zhou, zhy@ism.cams.cn Aiping Wu, wap@ism.cams.cn
Received Date: 15 July 2026
Accepted Date: 03 October 2026

Abstract

Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization worldwide. Its molecular epidemiology rests on consensus G-gene sequences, which capture only the dominant virus in each infection. Deep sequencing reveals far more variation inside each host. Linking those variants to one another gives a finer view of transmission. Here we reconstructed G-gene haplotypes from two independent RSV-A deep-sequencing datasets (Australia, n = 37; United States, n = 72). We phased variants using read-level linkage, then placed each haplotype on a global phylogeny. More than 90% of samples carry multiple haplotypes, which often sit on distinct phylogenetic branches. One cross-branch pair dominates each dataset: C4+C6 in 23 of 37 Australian samples (62%), and C18 + C21 in 26 of 72 US samples (36%). Both pairs recur far more often than month- and region-matched permutations predict (both P = 5.0 × 10^-5). Mutation-probability calculations suggest that recurrent within-host de novo mutation is unlikely to fully account for these patterns, particularly for the five linked sites in C4+C6 (probability ≤5.36 × 10^-15). Repeated joint introduction of pre-existing haplotypes offers a simpler explanation, and transmission-chain sampling can now test it directly. All branch-defining sites mapped to G-HVR2. In the examined global consensus G-gene set, dominant-branch states appeared, while minor-branch combinations were not detected under our criteria. Haplotype-resolved analysis thus turns sub-consensus diversity into linked, interpretable epidemiological signal.
- Respiratory syncytial virus A (RSV-A)
- , G gene
- , Within-host diversity
- , Intrahost evolution
- , Haplotype reconstruction
- , Variant linkage

References
1. Agoti, C.N., Otieno, J.R., Gitahi, C.W., Cane, P.A., Nokes, D.J., 2014. Rapid Spread and Diversification of Respiratory Syncytial Virus Genotype ON1, Kenya. Emerg. Infect. Dis. 20, 950-959.
2. Aksamentov, I., Roemer, C., Hodcroft, E., Neher, R., 2021. Nextclade: clade assignment, mutation calling and quality control for viral genomes. JOSS 6, 3773.
3. Altan-Bonnet, N., Perales, C., Domingo, E., 2019. Extracellular vesicles: Vehicles of en bloc viral transmission. Virus Research 265, 143-149.
4. Andino, R., Domingo, E., 2015. Viral quasispecies. Virology 479-480, 46-51.
5. Balaban, M., Moshiri, N., Mai, U., Jia, X., Mirarab, S., 2019. TreeCluster: Clustering biological sequences using phylogenetic trees. PLoS ONE 14, e0221068.
6. Battles, M.B., McLellan, J.S., 2019. Respiratory syncytial virus entry and how to block it. Nat Rev Microbiol 17, 233-245.
7. Beerenwinkel, N., Gunthard, H.F., Roth, V., Metzner, K.J., 2012. Challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data. Frontiers in microbiology 3, 329.
8. Botosso, V.F., Zanotto, P.M.D.A., Ueda, M., Arruda, E., Gilio, A.E., Vieira, S.E., Stewien, K.E., Peret, T.C.T., Jamal, L.F., Pardini, M.I.D.M.C., et al., 2009. Positive Selection Results in Frequent Reversible Amino Acid Replacements in the G Protein Gene of Human Respiratory Syncytial Virus. PLoS Pathog 5, e1000254.
9. Braun, K.M., Moreno, G.K., Wagner, C., Accola, M.A., Rehrauer, W.M., Baker, D.A., Koelle, K., O’Connor, D.H., Bedford, T., Friedrich, T.C., et al., 2021. Acute SARS-CoV-2 infections harbor limited within-host diversity and transmit via tight transmission bottlenecks. PLoS Pathog 17, e1009849.
10. Cane, P.A., 1997. Analysis of linear epitopes recognised by the primary human antibody response to a variable region of the attachment (G) protein of respiratory syncytial virus. J. Med. Virol. 51, 297-304.
11. Chen, S., Zhou, Y., Chen, Y., Gu, J., 2018. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics 34, i884-i890.
12. Cifuentes-Munoz, N., Dutch, R.E., 2019. To assemble or not to assemble: The changing rules of pneumovirus transmission. Virus Res. 265, 68-73.
13. Collins, P.L., Melero, J.A., 2011. Progress in understanding and controlling respiratory syncytial virus: Still crazy after all these years. Virus Research 162, 80-99.
14. Danecek, P., Bonfield, J.K., Liddle, J., Marshall, J., Ohan, V., Pollard, M.O., Whitwham, A., Keane, T., McCarthy, S.A., Davies, R.M., et al., 2021. Twelve years of SAMtools and BCFtools. GigaScience 10, giab008.
15. Do, L.A.H., Wilm, A., Van Doorn, H.R., Lam, H.M., Sim, S., Sukumaran, R., Tran, A.T., Nguyen, B.H., Tran, T.T.L., Tran, Q.H., et al., 2015. Direct whole-genome deep-sequencing of human respiratory syncytial virus A and B from Vietnamese children identifies distinct patterns of inter- and intra-host evolution. Journal of General Virology 96, 3470-3483.
16. Domingo, E., Perales, C., 2019. Viral quasispecies. PLoS Genet 15, e1008271.
17. Domingo, E., Sheldon, J., Perales, C., 2012. Viral Quasispecies Evolution. Microbiol Mol Biol Rev 76, 159-216.
18. Duvvuri, V.R., Granados, A., Rosenfeld, P., Bahl, J., Eshaghi, A., Gubbay, J.B., 2015. Genetic diversity and evolutionary insights of respiratory syncytial virus A ON1 genotype: global and local transmission dynamics. Sci Rep 5, 14268.
19. Eshaghi, A., Duvvuri, V.R., Lai, R., Nadarajah, J.T., Li, A., Patel, S.N., Low, D.E., Gubbay, J.B., 2012. Genetic Variability of Human Respiratory Syncytial Virus A Strains Circulating in Ontario: A Novel Genotype with a 72 Nucleotide G Gene Duplication. PLoS ONE 7, e32807.
20. Falsey, A.R., Hennessey, P.A., Formica, M.A., Cox, C., Walsh, E.E., 2005. Respiratory syncytial virus infection in elderly and high-risk adults. New England Journal of Medicine 352, 1749-1759.
21. Gonzalez-Parra, G., De Ridder, F., Huntjens, D., Roymans, D., Ispas, G., Dobrovolny, H.M., 2018. A comparison of RSV and influenza in vitro kinetic parameters reveals differences in infecting time. PLoS ONE 13, e0192645.
22. Goya, S., Ruis, C., Neher, R.A., Meijer, A., Aziz, A., Hinrichs, A.S., von Gottberg, A., Roemer, C., Amoako, D.G., Acuna, D., et al., 2024. Standardized phylogenetic classification of human respiratory syncytial virus below the subgroup level. Emerging Infectious Diseases 30, 1631.
23. Grad, Y.H., Newman, R., Zody, M., Yang, X., Murphy, R., Qu, J., Malboeuf, C.M., Levin, J.Z., Lipsitch, M., DeVincenzo, J., 2014. Within-Host Whole-Genome Deep Sequencing and Diversity Analysis of Human Respiratory Syncytial Virus Infection Reveals Dynamics of Genomic Diversity in the Absence and Presence of Immune Pressure. J Virol 88, 7286-7293.
24. Hadfield, J., Megill, C., Bell, S.M., Huddleston, J., Potter, B., Callender, C., Sagulenko, P., Bedford, T., Neher, R.A., 2018. Nextstrain: real-time tracking of pathogen evolution. Bioinformatics 34, 4121-4123.
25. Hoang, D.T., Chernomor, O., Von Haeseler, A., Minh, B.Q., Vinh, L.S., 2018. UFBoot2: Improving the Ultrafast Bootstrap Approximation. Molecular Biology and Evolution 35, 518-522.
26. Kalyaanamoorthy, S., Minh, B.Q., Wong, T.K.F., Von Haeseler, A., Jermiin, L.S., 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 14, 587-589.
27. Katoh, K., Standley, D.M., 2013. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30, 772-780.
28. Knyazev, S., Tsyvina, V., Shankar, A., Melnyk, A., Artyomenko, A., Malygina, T., Porozov, Y.B., Campbell, E.M., Switzer, W.M., Skums, P., et al., 2021. Accurate assembly of minority viral haplotypes from next-generation sequencing through efficient noise reduction. Nucleic Acids Research 49, e102.
29. Langedijk, A.C., Bont, L.J., 2023. Respiratory syncytial virus infection and novel interventions. Nat Rev Microbiol 21, 734-749.
30. Langedijk, A.C., Harding, E.R., Konya, B., Vrancken, B., Lebbink, R.J., Evers, A., Willemsen, J., Lemey, P., Bont, L.J., 2022. A systematic review on global RSV genetic data: Identification of knowledge gaps. Reviews in Medical Virology 32, e2284.
31. Langedijk, A.C., Vrancken, B., Lebbink, R.J., Wilkins, D., Kelly, E.J., Baraldi, E., Mascarenas De Los Santos, A.H., Danilenko, D.M., Choi, E.H., Palomino, M.A., et al., 2024. The genomic evolutionary dynamics and global circulation patterns of respiratory syncytial virus. Nat Commun 15, 3083.
32. Langedijk, J.P.M., Schaaper, W.M.M., Meloen, R.H., Van Oirschot, J.T., 1996. Proposed three-dimensional model for the attachment protein G of respiratory syncytial virus. Journal of General Virology 77, 1249-1257.
33. Lau, J.W., Kim, Y.-I., Murphy, R., Newman, R., Yang, X., Zody, M., DeVincenzo, J., Grad, Y.H., 2017. Deep sequencing of RSV from an adult challenge study and from naturally infected infants reveals heterogeneous diversification dynamics. Virology 510, 289-296.
34. Li, H., 2013. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. arXiv preprint arXiv:1303.3997.
35. Li, Y., Wang, X., Blau, D.M., Caballero, M.T., Feikin, D.R., Gill, C.J., Madhi, S.A., Omer, S.B., Simoes, E.A.F., Campbell, H., et al., 2022. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in children younger than 5 years in 2019: a systematic analysis. The Lancet 399, 2047-2064.
36. Li, Ying, Zhang, J., Liu, H., Noman, M., Rizwan, M.A., Isiaka, I.D., Chen, H., Li, W., Zhao, N., Li, Yan, et al., 2026. Age-related pharyngeal microbiome and host transcriptomic signatures underlying fever responses in RSV bronchiolitis. Virologica Sinica 41, 574-585.
37. Lin, G.-L., Drysdale, S.B., Snape, M.D., O’Connor, D., Brown, A., MacIntyre-Cockett, G., Mellado-Gomez, E., De Cesare, M., Bonsall, D., Ansari, M.A., et al., 2021. Distinct patterns of within-host virus populations between two subgroups of human respiratory syncytial virus. Nat Commun 12, 5125.
38. Lythgoe, K.A., Hall, M., Ferretti, L., De Cesare, M., MacIntyre-Cockett, G., Trebes, A., Andersson, M., Otecko, N., Wise, E.L., Moore, N., et al., 2021. SARS-CoV-2 within-host diversity and transmission. Science 372, eabg0821.
39. McCrone, J.T., Woods, R.J., Martin, E.T., Malosh, R.E., Monto, A.S., Lauring, A.S., 2018. Stochastic processes constrain the within and between host evolution of influenza virus. eLife 7, e35962.
40. Mehedi, M., McCarty, T., Martin, S.E., Le Nouen, C., Buehler, E., Chen, Y.-C., Smelkinson, M., Ganesan, S., Fischer, E.R., Brock, L.G., et al., 2016. Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread. PLoS Pathog 12, e1006062.
41. Nguyen, L.-T., Schmidt, H.A., Von Haeseler, A., Minh, B.Q., 2015. IQ-TREE: A Fast and Effective Stochastic Algorithm for Estimating Maximum-Likelihood Phylogenies. Molecular Biology and Evolution 32, 268-274.
42. Obando-Pacheco, P., Justicia-Grande, A.J., Rivero-Calle, I., Rodriguez-Tenreiro, C., Sly, P., Ramilo, O., Mejias, A., Baraldi, E., Papadopoulos, N.G., Nair, H., et al., 2018. Respiratory Syncytial Virus Seasonality: A Global Overview. The Journal of Infectious Diseases 217, 1356-1364.
43. Pangesti, K.N.A., Abd El Ghany, M., Walsh, M.G., Kesson, A.M., Hill-Cawthorne, G.A., 2018. Molecular epidemiology of respiratory syncytial virus. Reviews in Medical Virology 28, e1968.
44. Ruckwardt, T.J., 2023. The road to approved vaccines for respiratory syncytial virus. npj Vaccines 8, 138.
45. Sanjuan, R., 2017. Collective Infectious Units in Viruses. Trends in Microbiology 25, 402-412.
46. Sanjuan, R., Domingo-Calap, P., 2016. Mechanisms of viral mutation. Cell. Mol. Life Sci. 73, 4433-4448.
47. Savic, M., Penders, Y., Shi, T., Branche, A., Pircon, J., 2023. Respiratory syncytial virus disease burden in adults aged 60 years and older in high-income countries: A systematic literature review and meta-analysis. Influenza Resp Viruses 17, e13031.
48. Shi, T., Denouel, A., Tietjen, A.K., Campbell, I., Moran, E., Li, X., Campbell, H., Demont, C., Nyawanda, B.O., Chu, H.Y., 2020. Global disease burden estimates of respiratory syncytial virus-associated acute respiratory infection in older adults in 2015: a systematic review and meta-analysis. The Journal of infectious diseases 222, S577-S583.
49. Shi, T., McAllister, D.A., O’Brien, K.L., Simoes, E.A.F., Madhi, S.A., Gessner, B.D., Polack, F.P., Balsells, E., Acacio, S., Aguayo, C., et al., 2017. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study. The Lancet 390, 946-958.
50. Sullender, W.M., 2000. Respiratory syncytial virus genetic and antigenic diversity. Clinical microbiology reviews 13, 1-15.
51. Trento, A., Galiano, M., Videla, C., Carballal, G., Garcia-Barreno, B., Melero, J.A., Palomo, C., 2003. Major changes in the G protein of human respiratory syncytial virus isolates introduced by a duplication of 60 nucleotides. Journal of General Virology 84, 3115-3120.
52. Walker, G.J., Foster, C.S.P., Sevendal, A., Domazetovska, A., Kamalakkannan, A., Williams, P.C.M., Kim, K.W., Condylios, A., Stelzer-Braid, S., Bartlett, A.W., et al., 2024. Clinical, Genomic, and Immunological Characterization of RSV Surge in Sydney, Australia, 2022. Pediatrics 153, e2023063667.
53. Wang, Y., Wang, D., Zhang, L., Sun, W., Zhang, Z., Chen, W., Zhu, A., Huang, Y., Xiao, F., Yao, J., et al., 2021. Intra-host variation and evolutionary dynamics of SARS-CoV-2 populations in COVID-19 patients. Genome Med 13, 30.
54. Wei, X., Gao, Q., Wang, Y., Gao, X., Kou, Z., Li, X., Xu, Y., 2025a. Intra-host variation and transmission dynamics of SARS-CoV-2 Omicron outbreaks in Shandong, China. mSphere 10, e00355-25.
55. Wei, X., Li, H., Sai, L., Song, L., Gao, X., Li, C., Zhang, X., Shen, Y., Li, S., Liu, L., et al., 2025b. Low level of viral intra-host diversity is associated with severe SFTS disease. bioRxiv 2025-04.
56. Wilm, A., Aw, P.P.K., Bertrand, D., Yeo, G.H.T., Ong, S.H., Wong, C.H., Khor, C.C., Petric, R., Hibberd, M.L., Nagarajan, N., 2012. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Research 40, 11189-11201.
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