Construction and Infection Analysis of the Full-length cDNA Clone of XJ-160 Virus,the First Sindbis Virus Isolated in China

We reported the full-length infectious cDNA clone of XJ-160 strain of Sindbis virus firstly isolated in China. The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA and cloned into plasmid derived from pBR322 under the control of a SP6 promoter and followed by a polyA tail behind 3 terminus of genomic cDNA. It was stably amplified in Escherichia coli DH5. RNA transcribed from the full-length cDNA clone was highly infectious, after transfection into BHK-21 cell, resulting in generation of recovered progeny virus with titre of 10~7-10~8PFU/ml.A silent nucleotide change was introduced into the cDNA clone to create a Xba I site (C to T at 8453nt) that is used as a genetic marker of this infectious clone. This genetic marker was retained in the genome of recovered progeny virus. The recovered virus was indistinguishable from the parental virus XJ-160 in the aspects of cytopathogenic effect, plaque morphology on BHK-21cells, viral antigenicity, in vitro growth characteristics in BHK-21 cells and the virulence in suckling mice. The stable infectious cDNA clone of XJ-160 strain, as an effective reverse genetics experimental system, will provide a valuable molecular biological tool to study the pathogenesis and replication of Sindbis virus and to develop the vector system of Alphavirus.