O型口蹄疫病毒CATHAY株持续感染BHK21细胞系的建立

Establishment of a BHK21 cell line persistently infected by the CATHAY strain of foot-and-mouth disease O virus

为研究口蹄疫病毒(FMDV)持续感染形成的分子机理,本研究利用氯化铵、利巴韦林、免疫阳性血清和低剂量病毒处理O型FMDV(CATHAY株)和BHK21细胞建立持续感染细胞系,采用RT-PCR、核酸测序、IFA和Western-blot方法筛选和验证持续感染细胞模型。结果显示,除了低剂量病毒感染方法能连续传代,且从第5~20代次细胞中扩增到与亲代毒株高度同源的FMDV VP1基因,并能检测到VP1蛋白外,其他方法由于对细胞具有明显毒性作用,未能建立持续感染细胞模型。笔者认为化学药物可能不适合所选病毒毒株建立持续感染模型,而利用低剂量病毒法建立FMDV持续感染细胞系是一种简单可行的方法。上述结果表明,本研究成功建立了FMDV持续感染BHK21细胞模型,这为研究FMDV与宿主的互作关系,阐明FMDV持续感染机理奠定了重要的理论基础。

To investigate the molecular mechanism of the persistent infection of foot-and-mouth disease virus(FMDV),this research established a persistently infected cell line with O-type FMDV(CATHAY strain)and BHK21 cells using ammonium chloride,ribavirin,immune positive serum,and lower titer of FMDV.The persistent infection cell lines were screened and verified by RT-PCR,nucleic acid sequencing,immunofluorescence assay(IFA),and Western blotting.The result showed that the BHK-21 cell lines with FMDV persistent infection have been established by infection of lower titer of FMDV,the cell lines with FMDV can be continuously passaged.FMDV VP1 gene can be detectable from the 5th to 20th generation cells by RT-PCR and the sequences were highly homologous to the parental.The FMDV VP1 protein can be detectable by IFA in persistently infected cells.However,the remaining methods fail to develop the cell model of FMDV persistent infection due to their obvious toxic effects on cells.Therefore,we believed that the chemical drugs used in this study may be not a suitable reagent to develop the cell model of FMDV persistent infection, and direct infection using lower titer of FMDV is a simpleand feasible method. Collectively, we successfully established BHK-21 cell lines with FMDV persistent infection, which willhelpful to investigate the interaction between FMDV and the host and clarifying the mechanism of FMDV persistent infection.