病毒学报

目的 对2019年采集自新疆维吾尔自治区喀什地区伽师县的蚊虫样本中分离到的病毒株XJ1968进行鉴定，并进行比对分析。方法 2019年7月至9月，在新疆维吾尔自治区喀什地区伽师县通过诱蚊灯与诱蚊诱卵器开展蚊虫捕捉工作。采集样本低温处理运输至实验室之后制备研磨液，分别接种于哺乳动物BHK-21及蚊虫C6/36两种细胞系进行病毒的分离培养，提取病毒RNA并合成cDNA，利用二代测序技术测定全基因组序列。后续分析采用BioEdit比对同源性，并通过MEGA11构建系统发育树。结果 本次共采集蚊虫112批次，总计6328只，种群构成以尖音库蚊为主(占比69.11%)，其次为里海伊蚊(占比30.89%)。实验获得1株在C6/36细胞中出现阳性病变的分离物，但其在BHK-21细胞中未引起细胞病变，该特征提示该分离物可能具有昆虫宿主特异性。系统发育分析显示，XJ1968分离株归属于Alphamesonivirus cavallyense种分支，与南定病毒(Nam Dinh virus, NDiV)亲缘关系最近，处于同一进化分支。序列比对表明，该毒株与2009年瑞典分离的NDiV(OTU1株)具有最高的核苷酸及氨基酸同源性(97.7%)。对病毒编码的8个主要蛋白(包括与病毒复制相关的3CL^PRO、RdRp、HEL1、ExoN、NMT、OMT及结构蛋白S和N)进行同源性分析，结果显示与OTU1株的关键复制酶蛋白(3CL^PRO、RdRp、HEL1、ExoN)氨基酸同源性达99.1%～99.3%。结论 经分子生物学鉴定，该阳性分离物XJ1968为新型昆虫套式病毒NDiV。这是我国新疆维吾尔自治区喀什地区伽师县首次分离到该病毒，丰富了该地区蚊媒病毒本底数据。

Objective This study aims to identify and conduct a comparative analysis of the virus strain XJ1968, which was isolated from mosquito samples collected in the Xinjiang Uygur Autonomous Region, China in 2019. Methods Mosquito collection was performed in Jiashi County, Kashgar Prefecture, Xinjiang Uygur Autonomous Region, China from July to September 2019, utilizing mosquito lamps and oviposition traps. The collected samples were transported at low temperatures to the laboratory and processed into homogenates. These were subsequently inoculated into BHK-21(mammalian) and C6/36(mosquito) cell lines for virus isolation. Viral RNA was extracted from the positive isolates to synthesize cDNA, and the full genome sequence was determined with next-generation sequencing(NGS). Subsequent analysis included homology comparison using BioEdit, and phylogenetic tree construction using MEGA11. Results A total of 6,328 mosquitoes were captured in 112 batches. Culex pipiens was the predominant species, accounting for 69.11% of the total, followed by Aedes caspius(30.89%). One positive isolate was obtained from the C6/36 cell culture. However, this strain did not cause cytopathic effects(CPE) on BHK-21 cells, suggesting insect host specificity. Phylogenetic analysis showed that the XJ1968 isolate belongs to the species Alphamesonivirus cavallyense, clustering within the same evolutionary branch as Nam Dinh virus(NDiV). Sequence alignment revealed that XJ1968 had the highest nucleotide and amino acid homology(97.7%) with the NDiV OTU1 strain isolated in Sweden in 2009. Analysis of the eight major viral proteins(including the virus replicationassociated proteins 3 CL^PRO, RdRp, HEL1, ExoN, NMT, OMT and the structural proteins S and N) showed that the key replicase proteins(RdRp, 3 CL^PRO, HEL1, ExoN) shared amino acid homologies of 99.1%-99.3% with OTU1. Conclusion Molecular identification confirmed that the positive isolate XJ1968 is a newly isolated insect-specific nidovirus, Nam Dinh virus(NDiV). This represents the first isolation of this virus in Xinjiang Uygur Autonomous Region, China, enriching the baseline data about mosquito-borne viruses in this area.