Pathogenicity Research Of Infectious Hematopoietic Necrosis Virus JRT Genotype And Immune Protection Analysis Of Rainbow Trout IFNa Against IHNV

Infectious hematopoietic necrosis virus（IHNV）is a highly pathogenic cold-water fish virus.It is also an important pathogen affecting the world aquaculture economy and leading to clinical disease and death of various salmon species.An outbreak of IHNV may result in an 80-100%loss in the salmon farm.IHNV currently has five genotypes,each has different epidemic areas.And the main popular genotype in China is JRT-type.Although a large number of researches have been invested in the prevention and treatment of IHNV infection,the pathogenesis of IHNV have not been fully elucidated,especially in different genotypes.In 2016,our laboratory isolated a JRT-type IHNV in Chengdu,Sichuan Province,named DY2016.By studying the pathological changes of rainbow trout caused by IHNV DY2016and collecting the interaction information of IHNV DY2016 and host based on transcriptomics will help to understand the replication and pathogenesis of JRT-type IHNV,and provide new strategy for the comprehensive prevention and control of JRT-type IHNV.Interferon（IFN）is the first defense line of innate immunity after virus invading,and is very conservative in the evolution of vertebrates.Although IFN has been well studied in humans,mice and other mammals,there is still little information about the function and application of rainbow trout IFN.Studying the role of rainbow trout IFNa in combating JRT-type IHNV infection will help to develop safe,efficient,novel antiviral agents and immunopotentiators to enhance the resistance of fish to JRT-type IHNV.This study learned the occurrence regularity and clinical characteristics of disease induced by IHNVDY2016 by artificially infecting juvenile rainbow trout.And the expression profile of m RNA of rainbow trout kidney tissue after IHNV DY2016 infection was determined by RNA-Seq technique（Illumina）.The aim is to conduct a preliminary investigation of the pathogenesis of JRT-type IHNV.Subsequently,we cloned the rainbow trout IFNa gene and analyzed the molecular structure,and obtained the rainbow trout recombinant IFNa（rtrIFNa）by prokaryotic expression technique.And the immunoprotected effect of rtrIFNa against IHNV DY2016 was analyzed in vitro and in vivo.The research results are as follows:1.Isolation,identification and evolution analysis of IHNV DY2016This chapter analyzed the cause of disease on rainbow trout in the farm of Dayi,Sichuan Province in 2016.Through clinical observation,histopathological analysis,cell isolation and identification by RT-PCR,we found that the pathogen causing the rainbow trout disease was IHNV and named DY2016.The full-length nucleotide sequence of the glycoprotein（G）gene of IHNV DY2016 was amplified by designing specific primers.Sequencing analysis was performed after TA cloning,and the obtained nucleotide sequence was uploaded to NCBI to obtain the gene accession number MN475923.A phylogenetic tree was constructed based on the IHNV G gene nucleotide sequence to assess the genetic correlation between DY2016and 47 strains of IHNV world isolates.The homology of DY2016 and Chinese isolates（published in NCBI）is 98.7%-99.3%.Therefore,DY2016 belong to a single line of the JRT genotype Nagano subgroup.The maximum nucleotide diversity between DY2016 and Korean/Japanese JRT genotype IHNV isolates were 5.7%and 4.7%,respectively.The maximum nucleotide diversity of JRT genotype isolates in Asia is 6.3%,and its diversity is about 2-3 times to that of other genotypes,indicating that the IHNV of JRT genotype has a relatively rapid evolution rate.2.Acute infection analysis of IHNV DY2016According to the Karber method,the median lethal concentration of IHNV DY2016 was calculated.The LC₅₀ is about 8.61×10²TCID₅₀/m L.The rainbow trout juveniles were injected intraperitoneally with IHNV DY2016 at a dose of 6.84×10³ TCID₅₀ and observed at room temperature at 16°C.After 24 hours of infection,the rainbow trout juveniles showed signs of depression,abdominal expansion,skin darkening and exophthalmia.The survival curve showed that rainbow trout began to die after 48 hours post infection,and the cumulative mortality rate reached 100%after one-week infection.After 4 days of infection,The contents of red blood cell and hemoglobin were reduced in the blood,and the level of total protein,albumin and globulin in serum were decreased,but the contents of creatinine,urea,total bile acid,aspartate aminotransferase（AST）,alanine aminotransferase（ALT）,alkaline phosphatase and glucose increased,serum potassium increased,and serum sodium decreased.The main parenchymatous organs of rainbow trout,including liver,spleen,kidney,head kidney,heart,gill,intestine and brain,were observed by Hematoxylin-eosin（HE）staining at 48h&96h after infection.It was found that IHNV DY2016 caused different degrees of pathological damage to various tissues.The main histopathological changes are cell necrosis and severe inflammatory reactions.The infected tissue was homogenized and then inoculated into the cells,and IHNV was successfully isolated again,suggesting that the IHNV infection was successfully modeled.3.Transcriptomics study of rainbow trout kidney with IHNV DY2016 infectionThe mRNA changes of rainbow trout kidney tissue after 24h of IHNV DY2016 infection were detected by Illumina Hi Seq Xten high-throughput sequencing technology.The results showed that after infection,a total of 2225 differentially expressed genes（DEGs）were obtained,in which 1217 DEGs were significantly up-regulated and 1008 DEGs were significantly down-regulated.GO enrichment analysis of DEGs showed that DEGs is mainly involved in signal transduction,immune response,cell metabolism and cell adhesion.Among them,the GO-BP enrichment-related signaling pathways of down-regulated genes include immune response,neutrophil chemotaxis,and leukocyte migration.The KEGG signaling pathway enrichment analysis showed that the up-regulated DEGs was mainly enriched in proteasome,necroptosis,Toll-like receptor signaling pathway,etc.Down-regulated DEGs major enriched pathways include the pentose phosphate pathway,cytokine-cytokine receptor interactions,and cell adhesion molecules.Among the many DEGs,the genes associated with the complement system and the adaptive immune system,as well as the hemoglobin subunit genes,were all down-regulated to varying degrees.Genes associated with the interferon system and the apoptotic system were up-regulated,especially interferon-stimulated genes（ISGs）.The expression changes of these genes during IHNV DY2016infection may play an important role in the process of viral infection,but the related mechanisms need depth study.Six DEGs were randomly selected for quantitative real-time PCR（q RT-PCR）validation,the expression profiles were consistent with those obtained by sequencing.4.Cloning,expression and molecular characterization of rainbow trout IFNa geneRefer to the rainbow trout type I interferon IFNa（Gen Bank accession number:AM489418）published in NCBI Gen Bank,using the Signal P4.1 tool to find and remove the signal peptide sequence,designing primers for PCR amplification of the IFNa functional region,and then the IFNa PCR product was cloned into a T vector and sequenced.The results showed that the cloned rainbow trout IFNa domain was 453 bp in length and predicted to encode 151amino acids.It is a hydrophilic protein with a theoretical isoelectric point of 9.12.Cloned IFNa has two potential glycosylation site and four potential phosphorylation sites.Compared with the IFNa1 protein（accession number CAM28541.1）and IFNa3 protein（accession number AAV39394.1）,the amino acid sequence identity was 94.0%and 98.0%,respectively.The sequencing result was uploaded to Gen Bank,and the gene accession number was MK408448.Based on the cloned rainbow trout IFNa,the p ET32a（+）-IFNa expression plasmid was constructed and transformed into E.coli BL21（DE3）to establish a stable recombinant IFNa prokaryotic expression system and protein purification system.The relative molecular mass of rtrIFNa protein was about 35 k D as determined by SDS-PAGE,and the protein concentration after dialysis renaturation was about 500μg/m L.5.Activity analysis of rtrIFNa against IHNV DY2016rtrIFNa was inoculated on the rainbow trout gonad cell line（RTG-2）in vitro,and found that rtrIFNa has a good anti-IHNV DY2016 biological activity in dose-dependent.At a working concentration of 0.1μg/m L,almost half of the cells are protected from the virus infection.The potency is about 1×10⁴U/mg.Based on the nuclear protein N gene of IHNV DY2016,q RT-PCR was used to detect the proliferation of the virus.The p MD19-N plasmid template showed a good linear relationship with C_T values in the range of0.6×10⁶～0.6×10¹copies/μL,and the minimum detectable amount was 6 copies/μL.The results showed that the copies of the viral N gene were strongly inhibited after the incubation of rtrIFNa.The relative expression of interferon-stimulated genes（ISGs）was analyzed by relative q RT-PCR after stimulating by rtrIFNa.It was found that rtrIFNa can significantly increase the transcriptional expression level of ISGs compared with the control group.This indicates that rtrIFNa has the same activity as endogenous IFNa.rtrIFNa was intraperitoneally injected into the rainbow trout at a dose of 100μg/fish.After 24h of immunization,challenge was performed using IHNV DY2016 at a dose of 6.84×10³ TCID₅₀.The results showed that the juveniles in the control group began to die after 2 days post infection,while the juveniles in the rtrIFNa-treated group began to die after 4 days post infection,and the relative protection rate of rtrIFNa reached 45%within 14 days.6.The anti-IHNV DY2016 immunoprotection of rtrIFNa in vivoThe rainbow trout was treated with rtrIFNa at a dose of 100μg/fish for 24 h.After 3 days infection,the liver,spleen,kidney and head kidney tissues of the juveniles were collected.The pathological damage of the tissues was analyzed by making HE sections.The results show that the use of rtrIFNa can effectively decrease the tissues damage caused by IHNV DY2016 infection.In addition,the determination of SOD activity and MDA content in the above tissues revealed that rtrIFNa can significantly increase the antioxidant capacity of SOD in liver,spleen,kidney and head kidney tissues,and inhibit the increase of MDA content.It indicated that rtrIFNa has certain protection and repair effects on cell damage.Quantification of the copy number of the IHNV-N gene revealed that IHNV has different proliferation in different tissues,the highest content in the kidney,followed by the spleen and head kidney,and finally the liver,indicating that IHNV has typical tissue phagocytosis.This is also consistent with the result that IHNV DY2016 caused different degrees histopathological damage on different tissues.After rtrIFNa treatment,the proliferation rate of IHNV DY2016 in various tissues was significantly reduced.In addition,the use of rtrIFNa can also effectively increase the expression levels of IFNa,CD4,Ig M,and MHCII,CD8,MHCI,IL2 and TNFαimmune genes in tissues and increase the concentration of anti-IHNV antibodies in serum.