Supplementary Material for: Early Evolution of Human Respiratory Syncytial Virus ON1 Strains: Analysis of the Diversity in the C-Terminal Hypervariable Region of Glycoprotein Gene within the First 3.5 Years since Their Detection

Objective: Characterization of the phylogeny and diversity of human respiratory syncytial virus (HRSV) genotype ON1 that occurred during its early evolution (within the first 3.5 years since the detection of the first ON1 strains). ON1 strains have a 72-nucleotide-long in-frame duplication within the second hypervariable domain of the glycoprotein gene (HVR2). Methods: All available HVR2 sequences of strains belonging to the ON1 genotype published prior to June 20, 2014 were collected. Multiple sequence alignments, phylogeny, phylogeography, sequence clustering and putative protein analyses were performed. Results: The worldwide spread and diversification of ON1 strains are presented. Only in a minority of ON1 strains do the two replicas remain identical, and various ON1 strains possess common differences between the first and the second copy (segments A and B). Mutations of the progenitor sequence were more frequent in segment B, a higher overall diversity on the protein level and more putative glycosylation sites exist in segment B, and, unlike in segment A, positive selection acts on that protein region. Conclusions: The fast spread of the novel HRSV genotype ON1 has been accompanied by its rapid concurrent diversification. Differences in variability of the two replicas within HVR2 were detected, with C-terminal replica being more variable.研究目标为解析人类呼吸道合胞病毒（human respiratory syncytial virus, HRSV）ON1基因型在其早期演化阶段（首株ON1毒株被检出后的3.5年内）的系统发育特征与多样性。ON1毒株在糖蛋白基因的第二高变区（HVR2）内携带一段长度为72个核苷酸的框内重复序列。研究方法：收集2014年6月20日前已发表的所有ON1基因型毒株的HVR2序列，并开展多序列比对、系统发育分析、系统地理学分析、序列聚类分析以及推定蛋白功能分析。研究结果：本研究展示了ON1毒株的全球传播与演化分化情况。仅少数ON1毒株的两个重复拷贝序列保持一致，多数毒株的两个拷贝（A段与B段）之间存在共有差异。祖先序列的突变在B段更为频发；B段在蛋白水平上整体多样性更高，且存在更多推定糖基化位点；与A段不同，B段对应的蛋白区域受到正向选择作用。研究结论：新型HRSV ON1基因型的快速传播伴随其同步快速演化分化。本研究检测到HVR2内两个重复拷贝的变异度存在差异，其中C端重复拷贝的变异程度更高。