Genotype Identification and Genetic Characteristics of HCoV⁃NL63 Strains prevalent in Shanghai

Objective To identify the genotype of HCoV-NL63 strains prevalent in Shanghai and analyze the genetic characteristics of viral genome.Methods HCoV-NL63 positive samples were collected from Shanghai acute respiratory infection surveillance system from 2019 to 2024, whole genome sequences were obtained by next generation sequencing. Phylogenetic analysis based on S gene, orf1ab gene as well as whole genome sequence was conducted respectively. Sequence homology analysis of the whole genome and amino acid mutation site analysis of S gene were also conducted.Results Seven whole genome sequences of HCoV⁃NL63 were obtained from 16 positive samples. Compared to Guangzhou strains obtained in 2018, amino acid sequence identity of five B2 strains in this study was 99.3%-99.8%, and the nucleotide sequence identity was 99.6%-99.9% . Compared to Fukushima strains obtained in 2023, amino acid sequence identity of two C2 strains in this study was 99.8%~99.9%, the nucleotide sequence identity 99.9%. Compared with the original strain NC005831, there were 44 amino acid mutation sites located on S gene of five B2 strains, among which E572A was in the RBD region. There were 15 amino acid mutation sites in the S gene of C3 subtype, with I507L and E572A in the RBD region and I507L as the signature mutation of C3 subtype that was closely related to viral virulence.Conclusion Seven HCoV-NL63 sequences obtained in this study covered B2 subtype and C3 subtype. Five B2 strains appears on the same branch of the phylogenetic tree with Guangzhou strains that detected in 2018, and two C3 strains are on the same branch with Fukushima strains detected in 2023.

研究目的：本研究旨在明确上海市流行的人类冠状病毒NL63（HCoV-NL63）毒株基因型，并解析其病毒全基因组的遗传特征。
研究方法：本研究从2019年至2024年上海市急性呼吸道感染监测系统中收集HCoV-NL63阳性样本，通过下一代测序（next generation sequencing）获取全基因组序列；分别基于S基因、开放读码框1ab（orf1ab）及全基因组序列开展系统发育分析，同时进行全基因组序列同源性分析及S基因氨基酸突变位点分析。
研究结果：本研究从16份阳性样本中成功获得7条HCoV-NL63全基因组序列。与2018年分离的广州株相比，本研究中的5株B2亚型毒株的氨基酸序列同源性为99.3%~99.8%，核苷酸序列同源性为99.6%~99.9%；与2023年分离的福岛株相比，本研究中的2株C2亚型毒株的氨基酸序列同源性为99.8%~99.9%，核苷酸序列同源性为99.9%。以NC005831为原始参照株，5株B2亚型毒株的S基因共存在44个氨基酸突变位点，其中E572A突变位于受体结合域（Receptor Binding Domain，RBD）区域。C3亚型毒株的S基因共存在15个氨基酸突变位点，其中I507L与E572A均位于RBD区域，且I507L为C3亚型的特征性突变位点，该突变与病毒毒力密切相关。
研究结论：本研究获取的7条HCoV-NL63序列涵盖B2亚型与C3亚型；5株B2亚型毒株与2018年检出的广州株处于系统发育树的同一分支，2株C3亚型毒株则与2023年检出的福岛株处于同一分支。