深圳市输入性寨卡病毒分离株的进化分析及生物学特性研究

Study on the genetic evolution and biological characteristics of Zika virus strains isolated from a suspected case imported from Cambodia into Shenzhen

目的对从深圳宝安国际机场口岸入境的1例自柬埔寨归国发热人员进行寨卡病毒实验室检测,研究寨卡病毒分离株的遗传进化和生物学特征,为寨卡病毒病的预防控制提供参考依据。方法采集患者血液、唾液和尿液样本,采用荧光RT-PCR方法进行寨卡病毒核酸检测;选用多种组织培养细胞分离病毒,分别观察病毒株的致病变效应、空斑形成及病毒滴度等,并对两株病毒基因序列进行分子遗传进化研究,分析输入性病例来源。结果荧光RT-PCR结果显示,该病例血清、唾液以及尿液样本寨卡病毒核酸阳性并持续一段时间。首次成功从唾液和尿液标本中分离到寨卡病毒株,将其分别命名为ZIKV/S/SZ1901和ZIKV/U/SZ1901。两分离株均可引起Vero细胞病变,不引起BHK-21细胞病变;均可在Vero细胞中形成空斑,滴度分别为3.4×10^(5) pfu/mL、1.4×103 pfu/mL。RT-PCR扩增出预期大小约10272 bp片段,测序结果表明该片段与ZIKV/Hu/Thai/KngSG/17-D501株相应序列的同源性为99.6%。系统进化树显示该病毒属亚洲谱系,与输入至我国的其它寨卡病毒株位于不同的进化分支。寨卡病毒编码区氨基酸位点分析提示,SZ1901毒株在结构基因的氨基酸位点(D683E、V763M、T777M)和非结构基因NS1基因的(A188V)变异与近几年流行株完全相同。结论根据患者流行病学史、临床表现和实验室检测结果,确诊该病例为深圳市从柬埔寨输入性寨卡病毒感染病例,且寨卡病毒分离株具备与2016年以来流行的寨卡病毒大部分相同的分子基础。

In this study,the genetic evolution and biological characteristics of Zika virus strains isolated from a suspected case imported from Cambodia into Shenzhen were analyzed,to provide information for preventing Zika virus infection.Serum,saliva and urine samples were collected from a suspected case returning from Cambodia in 2019.We detected ZIKV RNA using real-time fluorescence RT-PCR methods.The samples were further inoculated into Vero and BHK-21 cells for virus isolation.The cytopathic effects(CPEs),plaque formation and virus titration of the two strains of virus in various tissue culture cells were investigated,and the genetic sequences of the two strains of virus were analyzed by molecular genetic analysis,as well as exploring its possible origin.The real-time fluorescent RT-PCR result showed that saliva and urine were ZIKV RNA positive while serum was weakly positive,and this continued for a period of time.Zika virus strains were successfully isolated from saliva and urine samples and named ZIKV/S/SZ1901 and ZIKV/U/SZ1901,respectively.The results showed that the CPEs of Zika viruses isolated from saliva and urine samples were observed at 96 hours in Vero cells.However,they did not all induce CPEs in BHK-21 cells,even though viral genes were amplified.The amplification titers of the ZIKV/S/SZ1901 and ZIKV/U/SZ1901 strains in Vero cells were 3.4×10^(5) pfu/mL and 1.4×10^(3) pfu/mL,respectively.A specific 10272 bp fragment was amplified by RT-PCR.Sequence analysis showed 99.6%homology to the corresponding sequence of the Thailand ZIKV Strain ZIKV/Hu/Thai/KngSG/17-D501.The phylogenetic tree indicated that the SZ1901 strain belongs to the Asia lineage and is located in a different evolutionary branch from other Zika virus strains imported into China.Amino acid analyses of the coding region of Zika viruses indicated that the SZ1901 virus strain had the same variation in amino acid sites of structural genes(D683E,V763M,T777M)and the non-structural NS1 gene(A188V)as Zika virus strains commonly isolated in recent years.According to the results of the epidemiological investigation,the clinical manifestations and nucleic acid detection from cases,the suspected case was confirmed to be infected with Zika virus.The results indicated that the SZ1901 strain had a similar molecular basis to the Zika virus that has been prevalent since 2016.