乙脑病毒野毒株SA14感染性克隆的构建及病毒拯救

Construction of an infectious clone and rescue of the SA14 strain of wild-type Japanese encephalitis virus

目的构建乙脑病毒野毒株SA14感染性克隆并进行病毒拯救。方法分节段合成乙脑病毒野毒株SA14全长cDNA序列,采用分段连接方式构建SA14全长cDNA质粒,以其为模板体外转录RNA并电转染导入BHK21细胞进行病毒拯救,用噬斑试验和间接免疫荧光法鉴定拯救病毒;绘病毒生长曲线,测定拯救病毒对小鼠的神经毒力。结果酶切鉴定质粒模板成功构建,用免疫荧光法检测到恢复病毒包膜蛋白表达,噬斑实验发现乙脑野毒株噬斑直径大于疫苗株;绘制病毒生长曲线,野毒株和疫苗株均在60h达到高峰。用拯救病毒进行动物感染试验,野毒株对小鼠脑内神经毒力LD50为10-1.07,皮下神经毒力LD50为100.33。结论成功建立乙脑野毒株SA14感染性克隆并拯救病毒,为进一步研究乙脑病毒的致病机制等奠定了基础。

Objectives To construct an infectious clone and to rescue the SA14 strain of wild-type Japanese encephalitis virus（JEV）. Methods The synthesized full-length cDNA of JEV SA14 was cloned into the low-copy plasmid pACNR with DNA ligase,and the resulting plasmids were identified with restriction enzyme digestion.RNA was transcribed from the plasmid template in vitro and electrotransfected into BHK21 cells.The culture supernatant was collected,and rescued viruses were detected with a plaque assay.The envelope protein of rescued viruses was detected with an immunofluorescence assay.The growth curve of rescued viruses was analyzed,and neurovirulence was determined in Kunming mice.Results Restriction enzyme digestion indicated that an infectious clone containing the full-length cDNA of JEV SA14 was successfully constructed.The envelope protein of rescued viruses was recognized by monoclonal antibodies against the envelope protein of JEV according to an immunofluorescence assay.Analysis of the plaque sizes of rescued viruses revealed that those of SA14 were larger than those of SA14-14-2.The growth curve indicated that the viral titer of the SA14 strain and the SA14-14-2 strain peaked 60 hpost-inoculation.The LD50（log10 PFU）value of rescued viruses was 10-1.07 when injected intracranially and 100.33 when injected subcutaneously. Conclusion A plasmid containing the full-length cDNA of the SA14 strain of JEV was constructed,and the rescued viruses were successfully rescued.This work has laid the foundation for research into the virulence of the SA14 strain of JEV.