Pathogenicity And Complete Genome Analysis Of A New Genotype Avian Hepatitis E Virus Va Hev-hb Strain

Avian hepatitis E virus(aHEV),the causative agent of big liver and spleen(BLS)disease and hepatic rupture hemorrhage syndrome(HRHS),mainly infects 30-72 week old laying hens and broiler breeder hens and causes reds fluid in the abdomen,regressive ovaries,liver steatosis and amyloidosis,enlarged liver and spleen and the synthetically lethality rate was1%.Since 2016,hepatic rupture hemorrhage syndrome characterized with hepatomegaly and rupture was observed in many chicken flocks in China.Avian HEV were multiple identified in interrelated flocks and VaHEV-HB strain was isolated from a laying hen farm in Hebei Province.In this study,we demonstrated the strong pathogenicity of the VaHEV-HB strain and it can also cause serious illness among chicks compared to other aHEV reference strains.How is the highly pathogenic aHEV prevalenting in domestic poultry different from that reported aHEV in abroad? What the immunological responses induced in infected flocks? In this study,the pathogenicity of the strain was studied through systematic animal experiments,and the part innate immune response to VaHEV-HB infection was comprehensively assessed from real-time fluorescent quantitative PCR and transcriptome sequencing.Its whole genome was sequenced and analyzed to explore the molecular basis of increased pathogenicity and to provide insights into molecular variation and pathogenic mechanisms to understand the prevalence of avian HEV infection in China.In order to observe the pathogenicity of VaHEV-HB to SPF chicks,ninety one-day-old SPF chicks were randomly divided into three groups,which were challenged by oral and intravenous routes.The body weight,the injury of the spleen and liver and the levels of immune-associated cytokines of chicks after infection was statistically analyzed.The results show that VaHEV-HB infection can indeed cause obvious hepatosplenomegaly in chicks.However,from the results of immune surveillance,OASL and IL-6 were all up-regulated more than 10,000 times after infection,whereas the other cytokines remained unchanged,andTLR and IFN were found to be downregulated.And the transcriptome sequencing results were consistent with the results of real-time PCR,and no genes related to immune response had changed significantly.Therefore,we speculate that this may be the reason why avian HEV can escape the immune system and remain in the body for a long time.To further understand the characteristics of VaHEV-HB strain,genotype and molecular characterization has been intensively studied.The complete genome sequence of this strain was obtained by amplification,sequencing and splicing.The results showed that the full-length genome of VaHEV-HB strain is 6652 bp with 127 bp of 5'-NCR and 25 bp of3'-NCR.The nucleotide and amino acid sequences from the VaHEV-HB strain was compared to 10 published references by DNAStar-Lasergene.Sequence analysis showed that VaHEV-HB shared 79.5 to 86.9% identities with other avian HEV strains published in GenBank.The highest homology with the non-pathogenic Avirulent strain in the United State(Genbank number:EF206691)is 86.7%,and the homology with CaHEV in Shandong,China,which was discovered in 2010,is 82.6%.Phylogenetic analyses showed that VaHEV-HB strain is distantly related to the other reference strain.This strain was in a separate branch from all the other epidemic strains emerged in China,which belonged to the new genotype.In conclusion,an animal experiment was conducted to observe the pathogenicity of VaHEV-HB in this study,and the part innate immune responses induced by VaHEV-HB were studied by real-time fluorescent quantitative PCR and transcriptome sequencing during this process.The genome-wide analysis was further carried out to provide insights into molecular variation and pathogenic mechanisms to understand the prevalence of avian HEV infection in China.