Comparative Study On Transcriptome,SOCS3-Related Immune Pathway And Cytokine Changes In The Host Infected With Duck Hepatitis Virus

Duck hepatitis A virus?DHAV-1?is one of the important pathogens that harm the duck industry.The attenuated vaccine CH60 strain is cultivated through serial passage on chicken embryos with DHAV-1 CH virulent strain and is widely used for prevention and control of the disease.However,the comparetive study on the host infected with DHAV-1 virulent strain and attenuated vaccine are still relatively lacking.This study explored this issue and the following results were obtained:1.Transcriptome sequencing and analysis of chicken embryo liver infected with DHAV-112-day-old SPF embryonated eggs were inoculated with DHAV-1 virulent strain?CH strain?and attenuated vaccine?CH60 strain?,and the embryonated eggs livers were collected at 12,24,36 and48h post infection?hpi?,and their pathological changes were analyzed.The liver from embryonated eggs at 48hpi was performed to transcriptome sequencing and transmission electron microscopy.The results showed that ecchymotic hemorrhage were observed in the livers of CH60-infected embryonated eggs at 48 hpi,which corresponded to histopathological lesions.The hepatic sinusoids were infiltratied by large numbers of red blood cells,which was accompanied by disordered hepatic cords.Additionally,parts of the cell nuclei underwent pyknosis,karyolysis,or karyorrhexis Meanwhile,transmission electron microscopic analysis showed that there was the exit of large numbers of lipoid drops from the hepatocytes,expansion of the rough endoplasmic reticulum,and swelling of the mitochondria where the ridge was fractured.There was no significant change in the hepatocytes of CH group except for the accumulation of large amounts of glycogen.Transcriptome sequencing analysis revealed that compared with the mock group,the CH and CH60 groups contained 150 and 2,336 DEGs,respectively?p-value<0.05 and fold change>2?.In addition,there were 2,191 DEGs between the CH60 and CH groups Furthermore,the infection with DHAV-1 strain CH60 is associated with enhanced type ? and II interferon responses,activated innate immune responses,elevated levels of suppressor of cytokine signaling 1 and 3?SOCS1 and SOCS3?accompanied with abnormalities in multiple metabolic pathways.Excessive inflammatory and innate immune responses induced by the CH60 strain are related to severe liver damage.The reliability of transcriptom data were further verified by qRT-PCR detection of DEGs?SOCS1/3,STAT1/3,IRF1/7,IFN-?/?,IL6 and TLR7?and SOCS3 mythylation detection.2.Effect of DHAV-1 on SOCS3-related immune pathwayTranscriptome sequencing has revealed that infection of chicken embryo livers with the CH60strain is associated with enhanced type ? and II interferon responses,activated innate immune responses,and abundant expression of SOCS1 and SOCS3.To explore the relationship between SOCS3-related immune pathway and viral replication,the following experiments were carried out.Chicken embryo fibroblasts?CEFs?were firstly infected with the DHAV-1 CH60 strain.The peak of viral proliferation occurred within 36-48 hpi(10^8.4copies/ml).The CH60 strain significantly induced the mRNA levels of IFN?,IFN?,SOCS3 and STAT1 in CEFs.To further study the effects of IFNs and SOCS3 on DHAV-1 virus replication,eukaryotic overexpression and shRNA interference were used,and SOCS3 overexpression significantly promoted viral replication,and knockdown SOCS3inhibited viral replication.Furthermore,SOCS3 overexpression significantly inhibited the expression of IFN?but promoted the expression of IFN?.To determine the effect of SOCS3-related immune pathways on DHAV-1 replication,the STAT1/3,MX1/OSAL and viral proliferation were examined via single and co-overexpression of SOCS3.SOCS3 overexpression clearly decreased the mRNA levels of STAT1/3 in the Janus kinase?JAK?-STAT signalling pathway,but co-overexpression of SOCS3 and IFN?/?could not inhibit the mRNA levels of STAT1/3,indicating that SOCS3 could not directly inhibit the transcription of STAT1/3 by inhibiting IFN?.The expression of MX1,but not OASL,was significantly inhibited after co-transfection of IFN?and SOCS3 into CEFs compared with that after co-transfection of IFN?,and the viral copy number was significantly upregulated compared with that in the IFN?and pCAGGS-co-transfected group.Subcellular localization of SOCS3 and IFN?revealed that SOCS3was mainly located in the nucleus and cytoplasm,while IFN?was located only in the cytoplasm.Co-localization of these two proteins was not observed in the cytoplasm.Immune-precipitation assays also indicated that SOCS3 and IFN?are not physically interacting.3.Cytokine changes in 7-day-old ducklings infected with DHAV-1 virulent and attenuated strains7-day-old ducklings were infected with DHAV-1 CH strain and CH60 strain,and visual observation and standard hematoxylin and eosin staining were performed to detect pathological damage in the liver,and viral copy numbers and cytokine expression in the liver were evaluated by quantitative PCR.The results showed that CH strain(10^8.4 copies/mg)had higher viral titers than the CH60 strain(10^4.9 copies/mg)in the liver and caused ecchymotic hemorrhaging on the liver surface.There were no obvious pathological changes in the CH60 group.Additionally,livers from ducklings inoculated with the CH strain were significantly infiltrated by numerous red blood cells,accompanied by severe cytokine storms,while the CH60 strain successfully induced type ? and type ?I interferons in the liver.The up-regulated expression of interleukins 6 and 10 stimulates moderate innate immunity and cellular immunity.In summary,the attenuated vaccine CH60 strain induces excessive inflammatory and innate immune responses which are related to severe liver damage and death of chicken embryonated eggs;inhibits the expression of IFN?by increasing the SOCS3 protein.SOCS3 can in turn decrease STAT1and STAT3 mRNA levels,thereby inhibiting the antiviral protein MX1 and ultimately promoting viral proliferation in the CEF;induces a moderate immune response to resist DHAV-1 virulent challenge in the ducklings.The mechanism of severe liver damage caused by virulent CH strain may be that it induces a strong cytokine storm in the liver,accompanied with numerous red blood cells infiltration and necrosis in the hepatocytes,resulting in serious morbidity and mortality of ducklings.