宁夏首例猴痘病毒测序及全基因组序列特征分析

Sequencing and Genomic Characterization of the first Monkeypox Virus in Ningxia,China

基于高通量测序技术和生物信息分析方法构建宁夏猴痘病毒分子溯源技术平台,为本地区猴痘疫情防控提供技术支撑。以宁夏首例猴痘病毒核酸阳性样本DNA为模板,超灵敏度猴痘病毒全基因组捕获构建扩增子测序文库,利用Illumina测序平台的Nextseq 2000进行高通量测序,序列拼接后使用在线分析工具Nextclade分析病毒核苷酸变异、基因组谱系分型并进行溯源。生物信息学分析中和抗体A35R蛋白序列及结构。口咽拭子和痘疱液靶基因F3L的Ct值分别为33.6和17.8,痘疱液的病载量高于咽拭子。痘疱液为样本测序后获得有效全基因组序列,测序平均深度为2306×,基因组覆盖率为99.12%,序列属于IIb(西非分支)C.1型,存在84个核苷酸突变位点,引起36个氨基酸突变。系统进化树分析结果显示,与GISAID猴痘病毒数据库中进化关系较近的为云南省提交序列EPI_ISL_18059183(2023⁃06⁃28采样)和日本近期的流行株,其中宁夏序列在共享81个核苷酸突变位点的基础上携带3个独有核苷酸突变位点(C94049T、C142797T、C174897T)。SWISS MODEL同源建模分析A35R蛋白的球状结构域与牛痘病毒A33R蛋白高度相似。成功从痘疱液中获得猴痘病毒全基因组序列,序列全长197209bp,构建了病毒变异和分子溯源技术平台。

A molecular tracing platform for the monkeypox virus was established by high⁃throughput sequencing and bioinformatics analysis.It was created to provide technical support for the prevention and control of monkeypox outbreaks in Ningxia,China.The DNA of the first positive sample of nucleic acids from the monkeypox virus was used as a template to construct an amplicon sequencing library for ultrasensitive capture of the whole genome.High⁃throughput sequencing was based on the Nextseq 2000 sequencing platform from Illumina.After sequence splicing,the online tools of Nextclade were used to analyze variations in the nucleotides,genome lineage typing,and traceability of the virus.Bioinformatics analysis was undertaken on the sequence and structure of the A35R protein of the neutralizing antibody.Ct values of the target gene F3L in oropharyngeal swabs and pustule fluid were 33.6 and 17.8,respectively,indicating a higher virus load in pustule fluid compared with oropharyngeal swabs.Whole⁃genome sequences were obtained from samples of pustule fluid with an average sequencing depth of 2,306×and genome coverage of 99.12%.The sequences belonged to type C.1 of clade IIb(West African branch)and contained 84 nucleotide⁃mutation sites,resulting in 36 amino⁃acid mutations.Analyses of phylogenetic trees showed that the sequences were closely related to EPI_ISL_18059183 submitted by Yunnan Province in China(sampled on 28 June 2023)and recent strains circulating in Japan.The Ningxia sequence carried three unique nucleotide⁃mutation sites(C94049T,C142797T,C174897T)in addition to sharing 81 nucleotide⁃mutation sites with these sequences.Homology modeling using SWISS MODEL revealed high similarity between the globular domain of A35R protein and that of A33R protein of the cowpox virus.The whole⁃genome sequence of the monkeypox virus was obtained from pustule fluid,and had a total length of 197,209 bp.A technical platform for mutations and molecular tracing of the monkeypox virus was also established.