The 164 K, 165 K, and 167 K residues of VP1 are vital for goose parvovirus proliferation in GEFs based on PCR-based reverse genetics system

Abstract

Background: Goose parvovirus (GPV) is the etiological agent of Derzsy's disease and is fatal for gosling. Research on the molecular basis of GPV pathogenicity has been hampered by the lack of a reliable reverse genetics system. At present, the GPV infectious clone has been rescued by transfection in the goose embryo, but the growth character of it is unclear in vitro. Methods: In this study, we identified the full-length genome of GPV RC16 from the clinical sample, which was cloned into the pACYC177, generating the pIRC16. The recombinant virus (rGPV RC16) was rescued by the transfection of pIRC16 into goose embryo fibroblasts (GEFs). The rescued virus was characterized by whole genome sequencing, indirect immunofluorescence assays (IFA) and western blot (WB) using rabbit anti-GPV Rep polyclonal antibody as the primary antibody. Previously, we found the 164 K, 165 K, and 167 K residues in the 160YPVVKKPKLTEE171 are required for the nuclear import of VP1 (Chen S, Liu P, He Y, et al. Virology 519:17-22). According to that, the GPV infectious clones with mutated K164A, K165A, or K167A in VP1 were constructed, rescued and passaged. Results: The rGPV RC16 has been successfully rescued by transfection of pIRC16 into the GEFs and can proliferate in vitro. Furthermore, the progeny virus produced by pIRC16 transfected cells was infectious in GEFs. Moreover, mutagenesis experiments showed that the rGPV RC16 with mutated 164 K, 165 K and 167 K in VP1 could not proliferate in GEFs based on the data of IFA and WB in parental virus and progeny virus. Conclusions: The rGPV RC16 containing genetic maker and the progeny virus are infectious in GEFs. The 164 K, 165 K, and 167 K of VP1 are vital for the proliferation of rGPV RC16 in vitro.