由构建的基因组全长cDNA所回收的我国登革2型病毒株的鉴定

Identification of the Chinese dengue 2virus strain rescued from the constructed full-length cDNA of viral genome

目的由构建的病毒基因组全长cDNA回收具有感染性的我国登革2型病毒株,为进一步阐明登革2型病毒的致病机制及其新型疫苗的研制奠定基础。方法用SP6RNA聚合酶系统制备我国登革2型病毒D2-43株基因组全长cDNA的体外RNA转录物,纯化后经电穿孔法转染C6/36细胞,观察致细胞病变作用以判断其感染性。从病变的细胞和培养上清中提取总RNA,通过RT-PCR扩增及克隆测序的方法证实细胞病变确为RNA转录物感染所致。同时收集可产生细胞病变的培养上清,再感染C6/36细胞以进一步证实所回收的登革2型病毒感染的稳定性。结果以我国D2-43病毒株基因组全长cDNA为模板制备的体外RNA转录物可使C6/36细胞产生病变,从病变细胞和培养上清中可扩增获得病毒特异的基因片段。在培养细胞中进行连续传代仍可产生细胞病变作用。结论构建的我国D2-43株基因组全长cDNA的体外RNA转录物对传代蚊细胞具有感染性,表明可产生完整的病毒颗粒。本研究可为阐明登革2型病毒的致病机理及探索新的预防和治疗措施奠定基础。

Aim To study the infectivity of the in vitro RNA transcript of the genomic full -length cDNA of Chin ese 43strain of dengue 2virus(D2-43),and hence to make some basis for elucidating the molecular pathogenesis of dengue virus and developing novel vaccines.Methods The in vitro RNA transcript of full -length cDNA o f the D2-43strain was prepared using the reagent and pr otocol supplied within the SP6RNA polymerase system.After purificat ion,it was transfected into C6/36ce ll line by electroporation.The infectivity of the D2-43virus,produced from the transfections of C6/36cells with the full -length RNA of viral genome,was determined by cytopathi c effect(CPE).Then the specific sequences of D2-43virus were amplif ied through reverse transcription PCR from the total RNA prepared from t he supernatant and infected C6/36cells,respectively,to verify th e specifity of the RNA transcript.For making sure the stability of the infe ctivity of RNA transcript,the cultu remedia were collected to conduct the s econd and third passages.Results The C6/36cells transfected with the in vitro RNA transcript can produce CPE,and specific sequences of D2-43can be obtained from the in-fected cells and culture media by RT -PCR amplification.Furthermore,the new virus from the RNA transcript can be passaged.Conclusion The in vitro RNA transcript prepared from the gen omic full -length cDNA of D2-43virus has specific and stable i nfectivity,demonstrating that the intact particles of dengue virus can be produced.The results obtained in the present study will shed some ligh t on elucidating the molecular mechanism of dengue virus pathogenesis and developing new prevention and therapy strategies against dengue -like diseases.