河南省4株新型冠状病毒的分离和病原特征分析

Analysis on the etiology and molecular epidemiological characteristics of SARS-CoV-2 in Henan Province

目的分离新型冠状病毒毒株,分析新型冠状病毒基本生物学特征和遗传进化特征。方法筛选具有代表性的新型冠状病毒核酸阳性样本,采用Vero和Vero-E6细胞系分离病毒、荧光定量RT-PCR方法初步鉴定,MiSeq测序技术获取新型冠状病毒全基因组序列,开展进化分析。结果4个新型冠状病毒核酸阳性的咽拭子样本在Vero和Vero E6细胞均产生细胞病变,细胞病变可以稳定传代。荧光定量RT-PCR鉴定四株病毒二代培养物均显示为新型冠状病毒核酸阳性;三株病毒二代培养物经Miseq测序获得新型冠状病毒全基因组序列,一株病毒二代培养物获得部分新型冠状病毒基因组序列。全基因序列比较显示三株新型冠状病毒均与武汉分离株Wuhan-Hu-1和WIV04亲缘关系最为接近,HN01与Wuhan-Hu-1株的NS3蛋白存在一个氨基酸差异,HN02与Wuhan-Hu-1株的NS8蛋白存在一个氨基酸差异,HN04与Wuhan-Hu-1株在同源编码区核苷酸同源性为100%。结论河南省新型冠状病毒株与湖北武汉株高度同源,暂未发现有意义的新型冠状病毒变异株。

Objective To isolate SARS-CoV-2(severe acute respiratory syndrom coronavirus 2)from throat swab samples of COVID-19(Coronavirus Disesae-2019)patients.To explore the basic biological and molecular epidemiological characteristics of SARS-CoV-2.Methods Vero and Vero-E6 cell lines were used for virus isolation.The real-time fluorescence quantitative RTPCR was used to identify SARS-CoV-2 preliminarily and MiSeq Sequencing System technology was used for the complete genome sequence of SARS-CoV-2 strains.Results Four SARS-CoV-2 nucleic acid positive throat swab samples procuded cytopathic effect on both Vero and Vero E6 cell lines,and cytopathic effect could stabilize passage.The four second generation strains were tested to be SARS-CoV-2 nucleic acid positive,three strains were complete genome sequenced.BLAST showed that all three SARS-CoV-2 strains we got were closest to Wuhan-Hu-1 and WIV04.There is an amino acid difference in NS3 protein between HN01 and Wuhan-Hu-1 strain,an amino acid difference in NS8 protein between HN02 and Wuhan-Hu-1.Conclusions Both Vero and Vero E6 cell lines could be used for isolation of SARS-CoV-2.The SARS-CoV-2 strains from Henan Province is highly homologous with strains from Wuhan.There was no meaningful SARS-CoV-2 mutant strain that has been found yet.