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

摘要:

【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 TCID₅₀/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。

关键词:猪δ冠状病毒;细胞系;易感性;感染;跨物种传播

Abstract:

[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 TCID₅₀/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.

Key words:porcine deltacoronavirus;cell lines;susceptibility;infection;cross-species transmission