不同动物源性细胞系对猪丁型冠状病毒的易感性

Susceptibility of cell lines from different animal species to porcine deltacoronavirus

【目的】探究猪丁型冠状病毒(porcine deltacoronavirus,PDCoV)能否在不同动物源性细胞系中感染和增殖。【方法】本研究将PDCoV四川分离株CHN-SC2015接种来自仓鼠、家禽、猴、人和猪的12种细胞系,将病毒在每种细胞系中盲传至少5代并通过逆转录-聚合酶链式反应(RT-PCR)、实时荧光定量PCR(RT-qPCR)、间接免疫荧光试验(IFA)和测序鉴定。【结果】PDCoV在Vero、PAM、PK15、ST和LLC-PK1细胞中的P1表现出明显的细胞病变效应(CPE),且在PAM、PK15、ST和LLC-PK1细胞上有不同程度的增殖,但在Vero和PAM细胞的后续传代中CPE逐渐消失;除3种易感细胞外(PK15、LLC-PK1和ST),病毒拷贝数随着传代次数的增加而逐渐降低,而在DEF、Marc-145、HEK-293、ZYM-SIEC02和PAM细胞的P4或P5中不能检测到PDCoV;PCR结果显示,只有在CEF和Vero细胞的P5中还能检测到PDCoV;IFA的结果表明,PDCoV可以感染除BHK-21和ZYM-SIEC02以外的大多数细胞,在PK15、LLC-PK1和ST细胞的P1、P3和P9均可以观察到特异性免疫荧光,因此仅3种细胞系(PK15、LLC-PK1和ST)适合病毒的连续传代,在P9病毒滴度分别可达10^(7.11)、10^(7.00)和10^(7.37) TCID_(50)/mL;在不同的细胞系中传代后,PDCoV的S基因序列累计有14个核苷酸和相应的12个氨基酸突变。【结论】本研究表明,PDCoV可在体外感染多种细胞,提示其可能存在跨种传播潜力。

[Objective]To explore whether porcine deltacoronavirus(PDCoV)can infect and proliferate in different animal species-derived cell lines.[Methods]The Sichuan isolate CHN-SC2015 of PDCoV was inoculated in twelve cell lines derived from hamster,poultry,monkey,human and swine.After at least five blindly passages in each cell line,the virus was identified by RT-PCR,RT-qPCR,indirect immunofluorescence assay(IFA),and sequencing.[Results]PDCoV caused distinct cytopathic effect(CPE)in Vero,PAM,PK15,ST,and LLC-PK1 cells at the 1st passage(P1)and proliferated to various degrees in PAM,PK15,ST,and LLC-PK1 cells,while the CPE gradually disappeared during subsequent passages in Vero and PAM cells.Except that in the three susceptible cell lines(PK15,LLC-PK1,and ST),the viral copies of the infected cell lines gradually decreased with the increase in passages,and PDCoV could not be detected at P4 or P5 of DEF,Marc-145,HEK-293,ZYM-SIEC02,and PAM cells.PCR results showed that PDCoV could be detected only in CEF and Vero cells at P5.The IFA results showed that PDCoV could infect other cell lines except BHK-21 and ZYM-SIEC02,and specific immunofluorescence was observed in PK15,LLC-PK1,and ST cells at P1,P3,and P9.Therefore,only three cell lines(PK15,LLC-PK1,and ST)were suitable for serial passage,with the virus titers up to 10^(7.11),10^(7.00),and 10^(7.37) TCID_(50)/mL at P9,respectively.After passage in different cell lines,CHN-SC2015 accumulated 14 nucleotide mutations corresponding to 12 amino acid mutations.[Conclusion]This study indicates that PDCoV can infect a variety of cells in vitro,suggesting that it may have the potential of cross-species transmission.