Construction of Infectious RNA Transcripts from Full-length cDNAs of Dengue 2 Viruse

Abstract:

To study the method of in vitro transcription and the infectivity of in vitro RNA transcripts of full-length cDNAs of dengue 2 viruse, thus to lay the foundation of constructing infectious dengue virus clone. The full-legnth cDNA of DEN2 NGC was amplified by long RT-PCR. With PCR products as templates, the in vitro RNA transcripts could be prepared by the SP6 RNA polymerase system. The transcripts were transfected into BHK-21 by electroporation and inoculated brain of suckling mice respectively. The effect of infectivity of the transcripts was observed. The specific sequences of DEN2 NGC strain were amplified through RT-PCR of total RNA extracted from brain of mice and infectious cells and the histopathologic changes of mice brain and cells were observed by electron microscope. The results indicated the specific fragment of the dengue virus could be amplified from infected mice brain and cells, and the virion could be observed. The in vitro RNA transcripts of full-length cDNAs of dengue virus were infectious. The dengue virus could be assembled by infecting brain of newborn mice and transfecting cell.