Isolation And Identification Of SVCV In Percocypris Pingi And Its Effect On I-IFNs Signaling Pathway

Spring viremia of carp is an acute,hemorrhagic,high-lethal infectious disease,it can infect variety of carp fish,and was recognized as one of the class-1 disease by animal epidemic prevention law of the people's republic of China.In 2016,a serious infectious disease with systemic multiple organ hemorrhage emerged in a Percocypris pingi farm of Sichuan Province,with a cumulative mortality rate of more than 40%,causing serious losses to farmers.In order to identify the etiology,this study isolated pathogens from the natural onset Percocypris pingi,and carried out histopathological observation,artificial infection test and semi-nested RT-PCR test to determine its pathogen as Spring viremia of Carp Virus(SVCV),at the same time,the genotype of glycoprotein gene was determined by constructing phylogenetic tree.The physico-chemica and biological characteristics of SVCV were determined,included virus morphology,virus titer,sensitivity to organic solvent,trypsin,acid,base,freezing and thawing times,temperature and suitable culture cell line,proliferation characteristics.Results showed that the morphology of the virus is typical of bullets,and sensitive to organic solvents,trypsin,acid and base,it can be inactivated at 60? for 30 minutes,but repeated freezing and thawing had little effect on its virulence.The optimum culture temperature was about 16?,the optimum cultured cells are EPC and FHM,and the virus titer(TCID50)is 107.24/0.1ml after culture 7d in EPC cell line.The virus begins to enter the logarithmic growth period at 24 h,and the virus titer reaches its peak and stabilizes at 96 h.In order to understand the effect of SVCV infection on the expression of host immune-related factors,this study conducted an in vitro experiment of SVCV infection with EPC cells as a model to detect the amount of toll-like receptors and immune-related factors including interferon regulatory factor(IRFs),interferon(IFNS),interferon stimulating genes(ISGS),and virus load at different points in time.Results showed that after infection with SVCV,the virus begins to replicate and proliferate at 6h,and the highest virus load at 96 h.In the early stage of infection,TLR3 and TLR4 transcription levels rose rapidly,at a maximum of 2.54 times and 79.67 times higher than the control group,respectively.Then,the immune response is activated,stimulated the production of IRF3 and IRF7,and then induced the expression of I-IFNs,in which the main role was IFNa1,and the highest expression was 2810.60 times of the control group.In the later stage of viral infection,I-IFNs stimulates the mass expression of ISGs,in which ISG15 and Vig-1 increased the most,with a maximum of 1889.96 times and 865.54 times of the control group,and then plays an anti-viral immune response effect,which indicated that these interferon immune-related factors are involved in the process of anti-SVCV of the host.