Study On The Infection Characteristics Of YT-aHEV Strain Of Avian Hepatitis E Virus And Its Mechanism Of Triggering Inflammatory Responses In Chickens

Hepatitis E virus（HEV）infection is a worldwide disease related to issues with public health,it has been reported in a number of animals in addition to humans.Avian HEV is the main causative agent of big liver and spleen（BLS）disease,or hepatitis-splenomegaly（HS）syndrome which has caused considerable economic loss in the poultry industry.Currently,several different genotypes of avian HEVs have been reported worldwide and epidemiological investigation revealed that avian HEV has been widespread in China in recent years.Due to the lack of efficient in vitro culture systems,molecular mechanisms underlying the HEV lifecycle are not known.In this study,we first isolated an cell-adapted strain of avian HEV from white-feathered broilers and focusing on the virus in vitro infection characteristics and in vivo pathogenicity.The study content includes the replication pattern of avian HEV in LMH cells,the pathogenicity of the isolate to SPF chickens and inflammatory response in vitro and in vivo induced by avian HEV infection of the host or host cells.This study is divided into three main sections.1、An aHEV strain was first isolated using an in vitro culture system.The samples of liver tissue were collected from white-feathered broilers in Shandong Province which displayed symptoms of enlargement and haemorrhage.The nucleic acid test of avian HEV showed that the liver tissue was positive.Virus isolation using LMH cells combined with indirect immunofluorescence（IF）analysis revealed an avian HEV strain was isolated from the liver tissue which was named YT-aHEV（YT strain）.To further confirm the isolation results,the YT strain was inoculated into LMH cells to amplify the virus in large quantities,and electron microscopy was used to confirm the presence of the concentrated and pruified virus particles which had typical aHEV characteristics.To further understand the molecular characteristics of the YT strain,the genome sequence of the YT strain was obtained using primer amplification,sequencing and splicing,the complete genome sequence homology between the YT strain and all genotype of aHEV in Genbank is 81.4～97.4%,according to the results of homology comparison and genetic evolution,the homology of the isolate is 97.4%similar to the CaHEV strain and segregated into the same branch belonging to avian HEV genotype 3.In summary,this study used LMH cells to isolate and identify an aHEV strain,setting the groundwork for future research into the pathogenicity and pathogenesis of the virus.2、Study on the in vitro infection characteristics and in vivo pathogenicity of YT-aHEV.To further determine the biological characteristics of the YT strain in vitro,the study first assessed the effect of YT strain infection on LMH cells.The finding demonstrated that the YT strain infection did not significantly alter cytopathic effect（CPE）,impair cell activity and proliferation,or significantly trigger cell apoptosis.The production of extracellular virus and intracellular virus was studied at the indicated times after infection:the copy number of intracellular viral RNA increased for the first 4 days after infection before starting to decline.The copy number of extracellular viral RNA increased after 6 days post-infection.The levels of infectious virus on ten consecutive passages on LMH cells determined by TCID₅₀had showed no significant rising trend.SPF chicken embryos were infected with YT strain through intravenous inoculation,chickens were necropsied at different time points after hatching,liver indices,liver lesions,virus isolation from various tissues organs,and histological changes were all calculated and recorded.The results demonstrated that the YT strain resulted in liver hemorrhage and swelling as well as growth suppression in chicken flocks.The results of viral isolation confirmed the liver was the predominant site of viral replication in vivo.Furthermore,in addition to liver,as well as in other organs like the spleen,where the virus was also present.3、YT strain triggered inflammatory response in vivo and in vitro.We hypothesize that one of the major pathogenesis-related elements for aHEV infection is the inflammatory response it causes based on clinical observations and artificial pathogenicity experiments.The host inflammatory response induced by the YT strain was observed in vivo and in vitro and the mechanisms of the inflammatory response were elucidated in this study.First,the expression of innate immune-related cytokines was examined both in vitro and vivo infections,the expression of OASL was up-regulated whereas the expression of MAVS was down-regulated.The levels of MX gene m RNA expression are also up-regulated by in vivo infection.Additionally in vivo and in vitro,ELISA was used to measure the levels of IL-1βand-18 secretion in the supernatants,IL-1βand-18 m RNA expression levers in the liver are detected using real-time q RT-PCR.Results demonstrated that the expression levels of IL-1βand-18 were significantly up-regulated after infection with YT strain in vitro and in vivo,indicating that the inflammatory response has been triggered.As an essential component of inflammatory responses and innate immunity,the m RNA expression of TLRs was determined by q RT-PCR at different time points following infection.The results showed that the m RNA expression levels of TLR3、4、15 and 21 were significantly up-regulated in infected LMH cells at 4 hpi.Viral-positive livers have the highest multiple changes in TLR3,followed by TLR4,TLR15 and TLR5,as compared to normal livers.Moreover,we examined the downstream signaling pathway of TLRs in LMH cells:the signaling pathways for NF-κB and MAPK are involved in the inflammatory response.LMH cells were pretreated with inhibitors of NF-κB and MAPK（JNK,p38,ERK1/2）signaling pathways for 2 h,respectively,then followed by viral infection.At 6 h post infection,the results showed that inhibition of NF-κB signaling pathway had no significant effect on m RNA levels of IL-1βand-18;IL-1βand-18 m RNA levels had significantly decreased after inhibition of p38 or ERK1/2 signaling pathway;also IL-18 m RNA levels had significantly decreased after inhibiting the JNK signaling pathway.The results were validated by IFA performed with IL-1βantibodies.The inflammasome complexes（inflammasome and caspase-1）plays a major role in the formation,maturation,and release of IL-1βand IL-18.The m RNA expression levels of NLRC3,NLRC5 and NLRX1were highly significantly up-regulated at 4 hpi,and both NLRP3 and NLPR1L were significantly up-regulated at 6 hpi（P<0.01）.The expression level of 5 inflammasomes in the livers were all up-regulated in YT-infected groups.Notably,NLRX1 was the gene with the most significantly up-regulated expression levels in both in vitro and in vivo experiments,WB results showed that NLRX1protein levels were significantly up-regulated in LMH cells 6hpi.The mitochondria of SPF chicken liver were damaged to some extent after viral infection,as demonstrated by transmission electron microscopy and mitochondrial fluorescence tests,and autophagy was also observed.Caspase-1 m RNA expression levels were up-regulated after infection in vitro and in vivo.When caspase-1 activity was inhibited using the inhibitor VX-765,the m RNA expression levels of IL-1β,IL-18 and several inflammasomes were also reduced.Moreover,it was found that the activity of MAPK and NF-κB signaling pathways interacts with inflammasomes.Virus copy numbers in different inhibitor treated LMH cells were determined and the results showed at 72 hpi,the VX-765 treatment group significantly inhibited intracellular viral replication（P<0.01）,while the SP600125 treatment group significantly inhibited intracellular viral replication（P<0.001）.The WB results showed that the JNK pathway was activated in both the early and late periods post-infection of LMH cells with the YT strain.In conclusion,an avian HEV strain that was cell-adapted was isolated for the first time in this study,and the LMH cell line allowed the virus to replicate steadily.Animal regression experiments confirmed that this strain caused liver damage and growth inhibition in chicken flocks.Infection with the YT strain led to host innate immunity in both in vivo and in vitro experiments,as well as a significant inflammatory response with reciprocal regulation of pertinent inflammatory pathways.