【摘要】 目的 分离在广西流行的新型冠状病毒（SARS-CoV-2）毒株并进行鉴定。方法 分别在2022年12月1-31日及2023年5月1-31日2个时间段，采集新型冠状病毒抗原试纸条检测阳性志愿者鼻、咽拭子标本，接种于VeroE6细胞，以细胞病变效应（CPE）作为评价指标，采用荧光定量PCR法检测出现CPE的细胞上清中SARS-CoV-2的开放阅读框1ab（ORF1ab）和核衣壳蛋白（N）基因。通过基因测序、病毒滴度测定等方法，明确所分离的毒株型别、S蛋白突变位点及病毒滴度。结果 共采集15份志愿者的鼻、咽拭子，接种于Vero-E6细胞4～5 d后，其中9例样本细胞出现CPE，形态为：细胞膨胀、脱落以及破裂死亡，镜下可见明显的合胞体。9例样本的ORF1ab和N基因均为阳性（CT值均<37）。使用Nanopore测序仪进行三代测序，运用MEGA软件构建最大似然法进化树显示本研究所分离的SARS-CoV-2毒株分别为BA.5.2.48、BF.7.14.1、XBB.1.9.1。S蛋白突变分析发现，部分毒株存在独特的氨基酸突变位点（G252A和C1243F）。分离获得的BA.5.2.48、BF.7.14.1、XBB.1.9.1毒株可在Vero-E6细胞上连续传代，病毒滴度测定结果显示，其细胞半数感染量分别为10^-5.67/100μL、10^-5.57/100μL、10^-4.5/100μL。BA.5.2.48与BF.7.14.1亚型毒株的样本接种于Vero-E6细胞后第2天可观察到轻微CPE，而XBB.1.9.1亚型毒株的样本接种细胞后第2天未观察到细胞出现明显变化。于接种后第4天所有待测样本陆续出现显著的CPE，于接种后第6天出现CPE的细胞比例可达75%以上。结论 成功分离并鉴定了3株SARS-CoV-2变异株，为开展毒株的基础研究工作奠定基础。更多还原

【Abstract】 Objective To isolate and characterize severe acute respiratory syndrome coronavirus 2（SARS-CoV-2） strain circulating in Guangxi. Methods During the periods of December 1-31, 2022 and May 1-31, 2023, nasopharyngeal and oropharyngeal swab specimens were collected from volunteers who tested positive by SARS-CoV-2 antigen rapid diagnostic tests. The specimens were inoculated into Vero-E6 cells, and viral isolation was evaluated based on cytopathic effect（CPE）. Viral RNA from CPE-positive supernatants was detected by quantitative reverse transcription PCR（qRT-PCR） targeting open reading frame 1 ab（ORF1 ab） and nucleocapsid（N） genes.The isolate’s strain type, S protein mutations, and viral titer are characterized by genetic sequencing and viral titer assays. Results A total of 15 nasal and pharyngeal swabs from suspected novel coronavirus-infected patients were collected and inoculated with Vero-E6 cells for 4-5 days, and in 9 of these samples, the cells showed CPE with the following morphology: cell swelling, detachment as well as rupture and death, and obvious syncytia were visible under the microscope. The ORF1 ab and N genes were detectable in all 9 samples（CT values <37）. The isolated strains were sequenced using Nanopore third-generation sequencing technology, and maximum likelihood phylogenetic trees was constructed with MEGA software identified the strains as BA.5.2.48, BF.7.14.1 and XBB.1.9.1. S-protein mutation analysis revealed the presence of unique amino acid mutation sites in some of the virulent strains（G252A and C1243F）. The isolated strains BA.5.2.48, BF.7.14.1 and XBB.1.9.1 were successfully passaged in VeroE6 cells, with tissue culture infective dose 50% determined to be 10^-5.67/100 μL, 10^-5.57/100 μL, 10^-4.5/100 μL, respectively. Mild CPE were observed as early as 2 days post-infection（dpi） in Vero-E6 cells inoculated with BA.5.2.48 and BF. 7.14.1, while no significant morphological changes were detected in XBB. 1.9.1-infected cells at this time point. All tested strains induced prominent CPE by 4 dpi, with more than 75% of cells exhibiting cytopathic changes by 6 dpi. Conclusion Three SARS-CoV-2 variant strains were successfully isolated and identified, laying the foundation for further basic research on these strains.更多还原