Study Molecular Basics Of IHNV Isolation And Infection

Infectious hematopoietic necrosis virus(IHNV)can cause most salmonoid fish acute and highly fatal disease,the diseased fish mainly manifests itself as maniac swims,Bleeding and necrosis of the kidney and spleen and other hematopoietic organs.The disease was first discovered in North America in the mid-20 th century for only a few decades and is have been widely spread around the world.The virus was also found in cold water fish in Northeast China in the 80 s of last century,the disease caused serious economic losses to salmon far ming around the world,and there are currently no effective vaccines and drugs,the MyD88/AP-1 pathway is mainly involved in cell proliferation,inflammatory response,embryo implantation and physiological and pathological pregnancy processes.Therefore,the experiment was carried out through the collection of diseased material from a cold water fish farm that was suspected of IHNV in Baishan City.The virus was isolated and identified by means of electron microscopy,salmon gonadal cell infection,virus gene extraction,cloning and sequencing.At the same time,we used healthy Salvelinus malma as an experimental animal and analysis of ploidy in healthy Salvelinus malma by flow cytometry,Selection of Salvelinus malma the same ploidy of isolation and identification of virus infection.After infection in 24 h,72h and 240 h molecular sampling of Salvelinus malma,collect the liver transcriptome sequencing.Analysis of sequencing results and screening for relevant gene inhibitors of MyD88/AP-1 signaling pathway to explore the response of this pathway to IHNV,and lays a theoretical foundation for the pathogenesis and replication of the virus of salmon family,provide effective support to the prevention and control of IHNV.The results were:1.The identification method and results of isolation of wild IHNV virus are as follows: Microscopically infecting rainbow trout gonad cells,cells began to develop lesions 36 hours later.At the same time,when the virus titer is 10-5.6,50% of the cells in the hole have lesions.The gene sequence analysis was used to establish the phylogenetic tree and found that the homology of the isolates was the highest with the HV7106 strain,reaching 97.01%.Symptoms of infection of the same ploidy Salvelinus malma were similar to those of fish under natural conditions.After 7 days of infection,the mortality rate reached 83%.2.Artificially infected salvelinus IHNV virus transcriptome sequencing results are analyzed as follows: Sequencing after transcriptome sequencing to see quality control,get a total of 94.05 Gb Clean Data,the percentage of Q30 bases in each sample is not less than 88.90%,indicating that the sequencing results are good.Select the BLAST parameter E-value not greater than 10-5 and the HMMER parameter not more than 10-10,and finally get 42315 Unigene with annotated information.IHNV challenged the experimental group of 240 h,namely S10,S11,S12 and the blank control group,that is,the differential genes in S1,S2,and S3 were classified using the COG database.The differential genes were selected,and the selected genes were verified by RT-PCR.Compared with the control group,the expression level of the above genes in the liver of the Salvelinus malma infected with IHNV was basically consistent with that of the transcriptome analysis.3.The effect of the MyD88/AP-1 pathway on the virus was analyzed as follows: Screening for the AP-1 inhibitor,nordihydroguaiaretic acid and acting on the AP-1 Toll-like receptor signaling pathway,real-time fluorescence quantitative PCR was used to design upstream and downstream primers.β-actin was an internal reference.The expression level of AP-1 gene was significantly down-regulated,reached the lowest value at 12 h,and was down-regulated by 1;at 24 h,the expression level increased.The expression level of P38 began to decrease at 3 h,decreased about 4 times at 12 h,and increased at 24 h,at 6h and 12 h and other treatment groups had significant difference.The expression of IL-1β was significantly decreased at the 12 th hour after NDGA treatment,and it decreased continuously within 12 h,there were significant differences between the other experimental groups and the 0h(P<0.05).