Pathogenicity Of Avian Hepatitis E Virus And Chicken Infectious Anemia Virus Co-infection In SPF Chickens

Avian hepatitis E virus(aHEV)is a hepatophilic virus that seriously harms the poultry industry and can lead to liver and spleen enlargement and even rupture in infected chickens,decreased egg production rate,and increased mortality rate.In recent years,epidemiological surveys have shown that this virus has been on the rise in China,there are also mixed infections with immunosuppressive viruses such as chicken infectious anemia virus(CIAV),avian leukemia virus(ALV),and chicken mariculosis virus(MDV).The co-infection pathogenicity of aHEV and ALV,aHEV and MDV has been observed in related studies,but there is a lack of research data on aHEV and CIAV co-infection pathogenicity.Firstly,CIAV and aHEV were inoculated by chicken embryo yolk sac inoculation and intravenous inoculation,and then infected with aHEV and CIAV after hatching to construct coinfection of aHEV and CIAV on SPF chickens,and experimental observation of co-infected groups and single-infected groups and blank control groups were conducted for 84 days.The pathogenicity of aHEV and CIAV co-infection in SPF chickens was studied by analyzing body weight gain,mortality,organ index,viral load in chickens at each infection stage,and pathological histological observations.The results showed that the mortality rates in ascending order were aHEV single-infected group,CIAV single-infected group,aHEV and CIAV coinfected group,and the co-infected group had the highest mortality rate.Body weight was consistently lowest in the aHEV and CIAV co-infected groups at all statistical time points,indicating that aHEV and CIAV co-infection exacerbated the suppressive effect of their respective single infections on body weight.Analysis of the developmental indices of each organ showed that the thymus developmental index was significantly lower in the CIAV singleinfected group compared with the blank control group(P < 0.05),and further reduced in the aHEV and CIAV co-infected group relative to the CIAV single-infected group.The liver development index was significantly increased in the aHEV and CIAV co-infected group relative to each single-infected group(P < 0.05),indicating that aHEV and CIAV co-infection can significantly increase liver enlargement.The pathological changes and histopathological analysis also showed that co-infection of aHEV and CIAV aggravated the pathogenic manifestations,resulting in severe atrophy of the thymus,hemorrhage,tissue structure destruction,and other pathological changes.Liver enlargement,hepatocyte necrosis,hepatic sinusoid expansion,and inflammatory cell infiltration.Spleen enlargement with grayish white lesions,decreased lymphocytes in the spleen,and proliferation of fibrous connective tissue.Large cyst of Fabricius.Secondly,in order to observe the interaction between aHEV and CIAV during the replication process in SPF chickens,the replication copy numbers of two types of viruses in the liver,spleen,thymus,and bursa of Fabricius in the co-infected and single-infected groups were detected by fluorescence quantitative PCR,and the differences in replication between the coinfected and single-infected viruses were compared with relative expression levels.The results showed that aHEV co-infection with CIAV promoted the replication of CIAV in four organs:liver,spleen,thymus,and bursa,as well as CIAV replication in blood.Conversely,co-infection of aHEV with CIAV promotes replication of aHEV in the liver,spleen,thymus,and bursa of Fabricius,as well as detoxification through the cloaca.To further observe the differences in aHEV and CIAV co-infection induced innate immune responses relative to their respective single infections,the relative expression of cytokines such as IL-2,IL-4,IL-6,IL-8,IL-10,IL-12,IFN-α,IFN-β,and IFN-γ in multiple organs at different time points was measured by fluorescence quantitative PCR in this study.Experiments have confirmed that co-infection with aHEV and CIAV can significantly increase the expression of IL-2 in the spleen,but inhibit its expression in the liver,and cause an upregulation of IL-4 expression in the liver and bursa of Fabricius,inhibiting the expression of IL-4 in the thymus.Co-infection significantly promotes the expression of IL-6,promotes the expression of IL-10 in the bursa of Fabricius,but inhibits the expression of IL-10 in the spleen.Co-infection with aHEV and CIAV promotes IFN-α and IFN-β expressed in the bursa of Fabricius.Immunosuppression is exacerbated by co-infection of aHEV with CIAV as opposed to aHEV and CIAV alone.In conclusion,this study successfully constructed an experimental animal model of coinfection of aHEV and CIAV in SPF chickens,and confirmed that the two aggravated the pathogenicity and immunosuppression in SPF chickens by promoting each other’s replication and changing cytokine expression levels during co-infection,suggesting that the detection and eradication of aHEV and CIAV should be strengthened.