我国登革2、3、4型病毒全长cDNA体外RNA转录体的感染性研究

Study on the infectivity of in vitro RNA transcripts from full-length cDNAs of dengue viruses type 2,3 and 4 isolated in China

目的 研究我国登革 2、3、4型病毒全长cDNA体外RNA转录体的感染性 ,为构建登革病毒的感染性克隆、深入阐明病毒的致病机理奠定基础。方法 以我国登革 2、3、4型病毒基因组全长cDNA为模板 ,用SP6RNA聚合酶系统制备体外RNA转录体 ,经电穿孔转染细胞 ,观察其致细胞病变效应。从病变细胞培养上清中提取总RNA ,用病毒特异引物进行RT PCR扩增 ,以证实细胞病变为转录体RNA的恢复病毒所致。结果 我国登革 2、3、4型病毒全长cDNA的体外RNA转录体均可使细胞产生病变 ,从细胞培养上清中可扩增出登革病毒特异的片段。结论 构建的我国登革 2、3、4型病毒全长cDNA的体外RNA转录体具有感染性 ,可在蚊传代细胞内恢复为登革病毒颗粒。

Objective To study the infectivity of in vitro RNA transcripts of full-length cDNAs of dengue viruses type 2, 3 and 4, thus to lay the foundation of constructing infectious dengue virus clone and in-depth elucidation of dengue pathogenesis. Methods Using the full-length cDNAs as templates, the in vitro RNA transcripts could be prepared by the SP6 RNA polymerase system. The RNA transcripts were then used to transfect BHK cells. Cytopathic effect (CPE) was observed to test the infectivity of the transcripts. Total RNA was extracted from the supernatant of the cells with typical CPE. Specific fragments of the dengue viruses were amplified by RT-PCR, testifying that CPE was caused by the recovered dengue viruses. Results The in vitro RNA transcripts of full-length cDNAs of dengue 2, 3 and 4 viruses could produce typical CPE in cultured cells, and the respective viral specific fragments of dengue could be amplified from the supernatant of the cell culture. Conclusion The in vitro RNA transcripts of full-length cDNAs of dengue viruses were infectious, and the dengue particles could be assembled from transcripts-transfected C6/36 cells.