Studies On Pathogenicity Of Co-Infection Of Avian Hepatitis E Virus And Marek’s Disease Virus

Avian hepatitis E virus（a HEV）causes a disease characterized by hepatitis-splenomegaly syndrome and big liver and spleen syndrome in chickens,mainly affects adult hens,resulting in increased mortality,reduced egg production,hepatosplenomegaly,vasculitis and hepatic amyloidosis,and the mortality rate is about 1%.The pathogenic characteristics and pathogenic mechanisms of a HEV are still unclear due to the lack of an effective cell culture system.Marek’s disease（MD）is a neoplastic disease caused by Marek’s disease virus（MDV）,which is widespread all over the world,also had an economically impact on the poultry industry worldwide.The disease can take several different,the most common form is lymphoproliferative disease which is produced by the tumors in various organs.The key measure to prevent MD in production practice is basically to prevent tumor through vaccine immunization.Although the use of vaccines has been successfully reduced loss which caused by the disease for a long time,MDV strains have shown continuous evolution in virulence,gaining the ability to overcome vaccine-induced immune responses.MDV can cause immunosuppression in infected chicken flocks,therefore it is commonly co-infected with other pathogens,such as avian leukosis virus（ALV）and reticuloendotheliosis virus（REV）,and it has become an important epidemiological factor contributing to the increased incidence rate of oncogenic diseases.The prevalence of hepatic rupture hemorrhagic syndrome（HRHS）had been occurred in laying hens and broilers in many provinces in China since 2016.Previous epidemiological data showed that there were mixed infections with multiple viruses,including a HEV,MDV,ALV,REV,fowl adenovirus（FAd V）,and chicken infectious anemia virus（CIAV）,suggesting that the occurrence of the disease might be associated with co-infection with multiple pathogens.In particular,several recent researches had been reported the co-infection of a HEV and MDV was detected in cases of hepatomegaly in chickens,suggesting that mixed infection with these two pathogens might be an important causative agents of liver rupture and exacerbation of liver hemorrhage in chickens.However,there is no report on the pathogenicity analysis of co-infections of a HEV and MDV.In this study,a HEV and MDV were simultaneously infected with Hy-line Brown laying hens and their pathogenicity was observed to further investigate the effect of co-infection with a HEV and MDV on the pathogenicity of chickens and its relevance with HRHS.To investigate the effect of co-infection of a HEV and MDV on virus replication,in this study the fluorescent quantitative PCR methods for a HEV and MDV detection were established to detect the viral loads of both viruses in vivo.Subsequently,200 Hy-line brown hens were grouped by designing the virus challenge experiment and controlling for multiple variables,chickens were necropsied and the samples of heart,liver,spleen,bursa and thymus were collected at 14,21,28,35,42 and 49 days post-infections.At the same time,the changes of body weight and immune organ index at different time points were recorded.The detection of the viral load and the duration of virus shedding in plasma at multiple time points by the established assay of FQ-PCR.The results of survival curve showed that the co-infection of a HEV and MDV had little effect on mortality rates,but the results of the body weight growth curve showed that the co-infection of a HEV and MDV had significant inhibition on the growth and development of chickens.In addition,necropsy showed that that the pathological changes of tissues and organs were more serious from the co-infection group,liver and spleen tissues showed obvious yellowing,enlargement and liver hemorrhage compared with the control group and the single-infection group.The histopathological observation showed that there were large areas of lymphocyte-like infiltration and congestion in the liver and spleen tissues from the co-infection group compared with the single-infection group.These results indicated that co-infection of the two viruses significantly enhanced the pathogenicity to infected chickens.The results of viral load assay showed that a HEV had no significant effect on the replication of MDV in the samples of blood and organs,however,MDV could inhibit the replication and shedding of a HEV,which might be related to the innate immune response induced by MDV infection,thus inhibiting a HEV replication.The results indicated that the co-infection of a HEV and MDV was part of the causative agents of clinical hepatic rupture hemorrhage syndrome.This study provided scientific data for exploring the pathogenic changes of a HEV and MDV during co-infection and their interactions in vivo,and provides new insights into the causes of "hepatic rupture and bleeding syndrome".