A novel porcine parvovirus DNA-launched infectious clone carrying stable double labels as an effective genetic platform

摘要

Porcine parvovirus (PPV) is one of the major pathogens causing reproductive failure of swine. However, its specific pathogenesis has not been fully elucidated. Infectious clone is a powerful tool for further studying the pathogenic mechanism of PPV. In the present study, a PPV infectious clone was constructed, and the clone carries His-tag and Flag-tag double-genetic marker at the end of the ns1 gene 3' terminal and vp1 gene 5' terminal, respectively. The PPV DNA fragment F1 (1-182) in 5' end and the other PPV DNA fragment F2 (47885074) in 3' end were synthesized and assembled to the lower copy plasmid to construct pKOLL(F1 + F2), while the PPV DNA genome as a template to amplify carrying tags sequence PPV middle DNA fragment F3 and F4 by introducing Flag and His tags sequence in primers. Subsequently, the fused fragment F3/F4 were cloned into the Stu I/Sna B I sites of pKQLL(F1 + F2) plasmid to assemble the complete full-length PPV DNA recombinant plasmids, named as pD-PPV. The pD-PPV was transfected into PK-15 cells to gain rescued PPV virus, designed as D-PPV. Moreover, D-PPV showed similar replicate capability and pathogenicity comparing to the wild-type parental PPV through in vitro and in vivo studies, and the double labels can effectively indicate the expression and localization of viral proteins. Finally, the rescued D-PPV was found to be a convenient tool for antiviral drug screening. These data indicated that the newly established reverse genetic system for PPV would be a useful tool for further studying the pathogenesis mechanisms of PPV, developing labeled vaccine and screening antiviral drug.