Genome Sequence Analysis And Establishment Of A Reverse Genetics System On An Infectious Pancreatic Necrosis Virus

Infectious pancreatic necrosis(IPN)is a major disease of salmon and trout seedlings,caused by infectious pancreatic necrosis virus(IPNV).There is no effective drug against IPN since introduced to China in 1980 s.Commercial IPNV vaccine cannot be introduced and applied directly in China due to mutations of virus trains and safety concerns,An IPNV isolate,designed ChRtm213,was isolated from a rainbow trout farming in Yunnan province of China in 2013.Complete genome sequence and molecular characteristics of the ChRtm213 was identified in this study.Furthermore,a reverse genetic system was performed to rescue recombinant IPNV.This study will benefit the comprehensive study of IPNV and reveal interaction mechanism between IPNV and host.Total RNA was extracted from cell cultures of IPNV-ChRtm213.The cDNA of IPNV complete gene was amplified by reverse transcription polymerase chain reaction(RT-PCR)strategy.Full genome sequence of the ChRtm213 was obtained and compared with the sequences of IPNV in GenBank.The results of sequence analyses showed that the complete genome sequence of ChRtm213 strain contains a segment A(3099 nucleotides)coding a polyprotein VP2-VP4-VP3,and a segment B(2789 nucleotides)coding a RNA-dependent RNA polymerase VP1.The phylogenetic analyses showed that ChRtm213 strain fell within genogroup 1,serotype A9(Jasper),having similarities of 96.3%(segment A)and 97.3%(segment B)with the IPNV strain AM98 from Japan.The results suggest that the Chinese IPNV isolate has relative closer relationship with Japanese IPNV strains.Efficient T7 RNA polymerase mediated reverse genetics system on IPNV was successfully constructed using an In-Fusion seamless connection method.Silencemolecular tags were introduced to segment A and B,and both modified genome segments were inserted into eukaryotic expression vector(designed NDV-vector)to construct recombinant plasmid ChRtm213-NDV-A and ChRtm213-NDV-B.Then the construct recombinant plasmids were co-transfected with plasmid expressing T7 RNA polymerase into CHSE-214 cells by LipofectamineTM2000(Invitrogen).The transfected CHSE-214 was subsequently inoculated to new monolayer of CHSE-214 at 96 h post transfection.Rescued recombinant IPNV was identified by plaque assay,electron microscope,indirect immunofluorescence test,enzyme molecular tag tests.In this study,an efficient reverse genetics system on IPN has been successfully established.The constructed system is stable and simple to produce recombinant IPNV on CHSE-214 efficiently.