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 (4788-5074) in 3' end were synthesized and assembled to the lower copy plasmid to construct pKQLL(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.,Copyright © 2019 Elsevier B.V. All rights reserved.