塞内卡病毒A型反向遗传株的快速构建OA

Senecavirus A(SVA)is an important pathogen of emerging swine idiopathic vesicular dis-ease,posing a serious threat to the healthy and stable development of the pig industry.Reverse genet-ics technology for RNA viruses is an indispensable tool for dissecting viral replication mechanisms,elucidating pathogenesis,exploring the functions of key genes,and developing novel vaccines.To over-come the relative complexity and long experimental cycle associated with the construction of conven-tional full-length infectious cDNA clones,a rapid rescue method for SVA based on an infections subge-nomic amplicon strategy was developed,without the need for in vitro ligation or bacterial cloning.Based on the whole-genome sequence of the SVA HBWH/1/2018 strain,two pairs of specific primers were de-signed to amplify the entire viral genome.A T7 promoter sequence was introduced into the 5′end of the upstream primer for fragment 1(F1),and a poly(A)tail was added to the 3′end of the downstream primer for fragment 2(F2).Two target fragments covering the full-length viral genome were obtained by high-fideli-ty RT-PCR amplification.The purified fragments were co-transfected into BSR-T7 cells,which stably ex-press T7 RNA polymerase.The supernatant was collected at 48 hours post-transfection and serially pas-saged for virus amplification.The rescued virus was identified by RT-PCR,sequencing,indirect im-munofluorescence assay,and electron microscopy.Viral biological characteristics were further ana-lyzed by plaque assay and one-step growth curve analysis.The results showed that SVA was successfully rescued in vitro,and the rescued virus exhibited replication kinetics and plaque phenotypes similar to those of the parental virus.This study established a rapid rescue method for SVA based on the overlap-ping subgenomic amplicon strategy.Compared with the conventional full-length infectious cDNA clone approach,this method features a simpler workflow and a markedly shortened experimental cycle.There-fore,this study provides a highly efficient and convenient technical platform for research on SVA pathogenesis,gene function analysis,and the development of novel vaccines.