我国登革2型病毒43株基因组全长cDNA体外RNA转录物感染性的研究

Study on the infectivity of the in vitro RNA transcript from genomic full-length cDNA of Chinese dengue 2 virus strain 43

目的 :研究我国登革 2型病毒 4 3株 (D2 4 3)基因组全长cDNA体外RNA转录物的感染性 ,为进一步阐明登革 2型病毒的致病机制及探索其新型疫苗奠定基础。方法 :用SP6RNA聚合酶系统制备D2 4 3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细胞以进一步证实该体外RNA转录物感染的稳定性。结果 :以我国D2 4 3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 4 3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型病毒的致病机制及探索新的预防和治疗措施奠定基础。

Objective: To study the infectivity of the in vitro RNA transcript of the genomic full length cDNA of strain 43 of Chinese dengue 2 virus (D2 43), and hence to provide the basis for elucidating the molecular pathogenesis of dengue virus and developing novel vaccines. Methods:The in vitro RNA transcript of full length cDNA of the D2 43 strain was prepared using the reagent and protocol supplied within the SP6 RNA polymerase system. After purification, it was used to transfect C6/36 cell line by electroporation. The infectivity of the D2 43 virus produced from the transfections of C6/36 cell with the full length RNA of viral genome was determined by cytopathic effect (CPE). Then the specific sequences of D2 43 virus were amplified through reverse transcription PCR of the total RNA prepared from the infected C6/36 cells and the supernatant of the culture, respectively, to verify the specificity of the RNA transcript. For making sure the stability of the infectivity of RNA transcript, the culture media were collected to conduct the second and third passages. Results: The C6/36 cells transfected with the in vitro RNA transcript showed CPE, and specific sequences of D2 43 could be obtained from the infected cells and culture media by RT PCR amplification. Furthermore, the new virus from the RNA transcript could be passaged. Conclusions: The in vitro RNA transcript prepared from the genomic full length cDNA of D2 43 virus has specific and stable infectivity, demonstrating that the intact dengue virus can be produced. The results obtained in the present study will shed some light on elucidating the molecular pathogenesis of dengue virus and developing new prevention and therapy strategies against dengue like diseases. [