Genomic Sequence Analysis And Establishment Of Reverse Genetics System For Atypical Porcine Pestivirus

Atypical porcine pestivirus(APPV)is a newly emerged virus which caused congenital tremor(CT)in newborn piglets,belonging to the genus Pestivirus of the family Flaviviridae.The piglets with CT are mainly characterized by muscle trembling,mobility limitation,and often died of insufficient sucking.APPV has been widely spread around the world since it was reported,bringing huge economic losses to the pig industry.To date,there are still lack of effective prevention measures for this virus.Here,we isolated the APPV CH-GD2017 strain and analyzed its whole genome sequence.Based on these results,we established the reverse genetics system of APPV for the first time,which provided basic data for studying the replication,assembly and invasion mechanism of APPV,and provided a feasible technical means and platform for the further development of APPV vaccines and the study of APPV molecular pathogenesis.In this study,the total RNA from cerebellar tissue of piglets with CT was used as the template for RT-PCR.The full-length genome of APPV CH-GD2017 strain was obtained by splicing the segmental sequences,and the sequence was submitted to Gen Bank,and the accession number was MK629522.The genetic evolution analysis of the APPV genome sequence was performed using biological software.The results showed that APPV CH-GD2017 strain had the highest homology with Chinese strains GD1,GD2 and GD3,while the lowest homology with Chinese GD-MH01-2018 strain,which were 99.4%?99.5%and 80.9%,respectively.The homology between members of the APPV in China,the USA,the Netherlands,Austria,Germany and Korea ranged between 83.0%and 83.5%.Further analysis of the E^rns,E2,NS3 and NS5B genes showed that the homologies between CH-GD2017 strain and members of the APPV in the USA,the Netherlands,Austria,Germany and Korea were 79.8%?83.7%,83.1%?86.3%,84.7%?86.3%and 82.4%?83.9%,respectively.Phylogenetic analysis showed that APPV CH-GD2017 strain was in the same branch as the Chinese strains GD1,GD2 and GD3,and belonged to the species Pestivirus K.And the CH-GD2017 strain formed independent branch from the USA,the Netherlands,Austria,Germany and Korea.In order to develop the reverse genetics system for APPV CH-GD2017 strain,six fragments of the CH-GD2017 strain genome were used as templates to generate the 5'and3'half-length fragments by Overlap PCR,and cloned into the p BR322 and p EGFP-N1vectors,respectively.Then the full-length fragment of APPV was obtained by Overlap PCR using the recombinant plasmids as templates.Subsequently,the infective viral RNA was obtained by in vitro transcription,and the RNA product was purified and transfected into PK-15 cells.At 72 h post-transfection,the rescued virus was harvested from the supernatant of transfected PK-15 cells by three freeze-thaw cycles.And the rescued virus was passaged ten times.It was proved that the APPV CH-GD2017 strain was rescued successfully after identified by RT-PCR and indirect immunofluorescent assay.To improve the efficiency of virus rescue,the obtained recombinant plasmids p BR-APPV5'and p EGFP-APPV3'were used as templates,and the full-length APPV fragment was ligated into the p BR322 vector to generate the full-length infectious clone of APPV by seamless cloning.The infectious clone was transfected into PK-15 cells,and APPV was successfully rescued.Our work contributed to understand the genetic evolution of APPV,enriched the APPV genome database,and also laid a solid foundation for exploring the structure and function of APPV genome,molecular pathogenesis and the development of novel vaccines.