Construction of a recombinant coxsackievirus and its infectivity

Objective To purify the coxsackievirus (CV) -A6 and CV-A16 cDNA, and construct a recombinant pBM16A-Re (CV-A16-CV-A6-VP) in which CV-A6-VP replaces CV-A16-VP, and to study its infectivity in order to provide references for the development of CV-A6, CV-A16 and recombinant vaccines. Methods Viral genomic RNA was extracted from CV-A6 and CV-A16 to obtain full-length cDNA by RT-PCR. The cDNA was purified and inserted into pBM16A-TOPO vector. Through T7 polymerase system the linear genomic cDNA was transcribed in vitro to obtain viral genomic RNA. After purification, this genomic RNA infected Vero cell to obtain a recombinant CV pBM16A-Re. The infectivity of the Re was detected by microscope observation, gene sequencing test and immunofluorescence. Results The electrophoresis results of CV-A6, CV-A16 and Re showed a band about 7 500 bp. Several targeted bands about 7 500 bp and a vector band about 1 800 bp were visible by restriction enzyme digestion of pBM16A-CV-A6, pBM16A-CV-A16 and pBM16A-Re. The constructed clones were consistent with the theoretical sequences by whole genome sequencing analysis. Morphological identification showed that cytopathic effect caused by typical enterovirus was observed in CV-A16 group, but not in CV-A6 and Re group. RT-PCR identification showed the same results. Conclusions The recombinant viruses of CV-A6, CV-A16 and Re are constructed successfully. The recombinant CV-A16 has the same infectivity as its wild type. The recombinant pBM16A-Re loses its cellular infectivity. Key words: Coxsackievirus infections; Reverse genetic technology; Recombinant virus; Infectivity