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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 in vitro RNA transcripts of the full-length cDNA of dengue 2 type 4 (D2 4 3) genome in China, and to lay the foundation for further clarifying the pathogenesis of dengue 2 virus and exploring its new vaccine. METHODS: In vitro RNA transcripts of D2 4 3 full length cDNA were prepared by SP6 RNA polymerase system. After purification, C6 / 36 cells were transfected by electroporation, and the cytopathic effect was observed to determine their infectivity. Total RNA was extracted from the diseased cells and culture supernatants. RT-PCR amplification and cloning sequencing confirmed that the cytopathic effect was indeed caused by RNA transcript infection. At the same time, the culture supernatant was collected to induce cytopathic effect and then infected with C6 / 36 cells to further confirm the stability of the in vitro RNA transcript infection. Results: In vitro RNA transcripts prepared from the full-length cDNA of D2 4 3 strain in China could induce the pathological changes of C6 / 36 cells. The virus-specific gene fragments were amplified from diseased cells and culture supernatant. Continuous passage in cultured cells can still produce cytopathic effects. CONCLUSION: In vitro RNA transcripts of full-length cDNA of D2 4 3 genomes constructed in China are infectious to mosquito-transmitted mosquitoes, indicating that they can produce complete virus particles. This study can lay the foundation for elucidating the pathogenesis of Dengue 2 virus and exploring new preventive and therapeutic measures.