HEV was first reported in Western Canada in 1991 and subsequently caused outbreak in the United States, Australia and the United Kingdom [8, 9]. It has been reported that severe HEV infections have occurred in several provinces in China since 2016 [10, 3, 11]. In recent years, outbreaks of BLS and HS syndrome in chicken flocks have gradually increased in Shandong province [10, 12]. In order to investigate the causes of BLS and HS syndrome, and the epidemic characteristics of avian HEV in Shandong province, 18 cases of hepatomegaly and splenomegaly were collected for histopathology examination and pathogen detection, including HEV, MDV, FAdV, ALV, CIAV, and REV. Histopathological examination found that hemorrhage and necrosis in the liver, the heterophagic granulocytes and lymphocytes were infiltrated in the portal areas of the liver, which were similar to the results of lymphocytic phlebitis and periphlebitis of the liver in the artificial challenge chickens infected with avian HEV [13, 1], moreover, the reduction of lymphocytes and amyloidosis were observed in the spleen. However, due to the lack of available cell for avian HEV isolation at present, the bile samples of HEV positive chickens instead of purified virus for animal challenge experiments [13, 1], therefore, the isolation of avian HEV strain and animal experiment to obtain the accuracy of the results will be the focus of our further research. Notably, there were a large number of heterophagic granulocytes infiltration in the liver in this study, this cell was related to bacterial infection, indicating that clinical avian HEV infections may have co-infection or secondary infections. The roles of the mixed infections of multiple viruses in BLS and HS syndrome cannot be ruled out.
Actually, it has been reported mixed infections of avian HEV and ALV [4], HEV and MDV since 2016 [14], and the mixed infection rate of avian HEV and several immunosuppressive viruses was up to 58% in chickens, China, including CIAV, ALV, and REV [5], which indicated that the mixed infections of HEV and other viruses were common in chicken flocks. In the current study, further PCR detection showed that all 11 HEV positive samples were mixed infections, of which the infection rates of HEV and CIAV, HEV and FAdV were highest. CIAV is an important immunosuppressive virus in chicken, and according to the epidemiology investigation of co-infection of vertically transmitted or immunosuppressive viruses in chickens by Li et al, CIAV had the highest detection ratio [15]. FAdV has been prevalent in poultry in recent years and caused huge economic losses to poultry industry in China [16, 17]. It has been reported that chickens infected with FAdV were more likely to be infected with immunosuppressive diseases [18], and previous study has also reported that the co-infection rate of HEV and FAdV was about 10% [5]. The problem of vaccine contaminated with CIAV and FAdV may facilitate to the serious mixed infection status [19]. However, what role have CIAV and FAdV played in the pathogenesis of avian HEV and whether they can promote the onset of HEV, these questions are worthy of further study. On the whole, this study further confirmed the pervasiveness and severity of avian HEV and other viruses in chicken flock, and the continuous epidemiological surveillance is required.
Different genotypes of avian HEV have been reported worldwide [10, 20, 21, 22]. Zhao et al. reported the complete genome sequence of the first avian HEV in 2010[3], since then, the avian HEV isolates of different genotypes have been widely reported in China, including Shandong, Jiangxi and Guangdong provinces [10, 13, 23]. In order to further determine the molecular characteristics of the 11 HEV isolates obtained in this study, nucleotide homology analysis was performed, it was found that the 11 HEV isolates belonged to Orthohepevirus B (75.9–83.7%), and genetic evolution analysis showed that all isolates located in a single branch, which was different from the known 4 genotypes strains, indicating they were a novel genotype. Through these results, we speculated that avian HEV may have been widespread in China, and the genetic background of HEV has become more and more complicated.