基孔肯雅病毒感染性克隆的构建及鉴定

Construction and identification of chikungunya virus infectious clones

目的构建基孔肯雅病毒(chikungunya virus,CHIKV)东中南非型Ross株及该型印度洋分支LR2006株的感染性克隆.方法分别将CHIKV Ross株和LR2006株基因组分段合成,构建完整基因组的转录质粒,转录制备病毒基因组RNA,转染Vero细胞,收集培养上清,接种Vero细胞扩增病毒,用扩增的病毒感染Huh7细胞,检测病毒蛋白的表达,经滴鼻途径将106 PFU病毒接种给C57BL/6小鼠,观察小鼠的体重改变以及存活情况.结果成功构建了2株CHIKV的全长基因组转录质粒,在RNA转染Vero细胞的上清中检测到感染性CHIKV.Ross株感染的5只小鼠在6天后体重下降(P<0.05),第11天小鼠全部死亡.LR2006株感染的5只小鼠仅1只死亡,且全部小鼠体重均无明显减轻.结论成功构建CHIKV的Ross株和LR2006株感染性克隆,Ross株对C57BL/6小鼠致病性强于LR2006株.

Objective To construct chikungunya virus(CHIKV)infectious clones based on the Ross strain of the Eastern-Central-Southern African(ECSA)genotype and the LR2006 strain of the Indian Ocean lineage(IOL).Methods The genome fragments of CHIKV Ross strain and LR2006 strains were separately synthesized.The complete genomic transcriptional plasmids were constructed.The genomic RNAs were prepared by transcription and capping,and then were transfected into Vero cells.The supernatant was collected and inoculated Vero cells to amplify the viruses.The amplified viruses were used to infect the Huh7 cells to detect the viral proteins expression.Each virus at the titer of 106 PFU was intranasally inoculated five C57BL/6 mice.The body weight changes and survival of the mice were observed.Results Full-length genomic transcription plasmids of two CHIKV strains were constructed.Infectious CHIKV could be detected in the supernatant of each viral genomic RNA transfected Vero cells.The mice infected with the recovered Ross strain showed a significant decrease in body weight from 6 days after infection(P<0.05)and all died on the 11th day.Only one of the mice infected with the LR2006 strain died,and all the mice had no significant body weight loss.Conclusions The infectious clones of CHIKV Ross strain and LR2006 strain were successfully constructed,and the pathogenicity of the Ross strain was stronger than that of the LR2006 strain in C57BL/6 mice.