Generation and Infectivity Titration of an Infectious Stock of Avian Hepatitis E Virus (HEV) in Chickens and Cross-Species Infection of Turkeys with Avian HEV

Abstract

Avian hepatitis E virus (HEV), a novel virus identified from chickens with hepatitis-splenomegaly syndrome in the United States, is genetically and antigenically related to human HEV. In order to further characterize avian HEV, an infectious viral stock with a known infectious titer must be generated, as HEV cannot be propagated in vitro. Bile and feces collected from specific-pathogen-free (SPF) chickens experimentally infected with avian HEV were used to prepare an avian HEV infectious stock as a 10% suspension of positive fecal and bile samples in phosphate-buffered saline. The infectivity titer of this infectious stock was determined by inoculating 1-week-old SPF chickens intravenously with 200 l of each of serial 10-fold dilutions (10 ؊2 to 10 ؊6 ) of the avian HEV stock (two chickens were inoculated with each dilution). All chickens inoculated with the 10 ؊2 to 10 ؊4 dilutions of the infectious stock and one of the two chickens inoculated with the 10 ؊5 dilution, but neither of the chickens inoculated with the 10 ؊6 dilution, became seropositive for anti-avian HEV antibody at 4 weeks postinoculation (wpi). Two serologically negative contact control chickens housed together with chickens inoculated with the 10 ؊2 dilution also seroconverted at 8 wpi. Viremia and shedding of virus in feces were variable in chickens inoculated with the 10 ؊2 to 10 ؊5 dilutions but were not detectable in those inoculated with the 10 ؊6 dilution. The infectivity titer of the infectious avian HEV stock was determined to be 5 ؋ 10 5 50% chicken infectious doses (CID 50 ) per ml. Eight 1-week-old turkeys were intravenously inoculated with 10 5 CID 50 of avian HEV, and another group of nine turkeys were not inoculated and were used as controls. The inoculated turkeys seroconverted at 4 to 8 wpi. In the inoculated turkeys, viremia was detected at 2 to 6 wpi and shedding of virus in feces was detected at 4 to 7 wpi. A serologically negative contact control turkey housed together with the inoculated ones also became infected through direct contact. This is the first demonstration of cross-species infection by avian HEV.Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important human pathogen (1-2, 23-24, 26, 34-35). HEV is a positive-sense, single-stranded, nonenveloped RNA virus. The genome of HEV is about 7.2 kb and contains three open reading frames (ORFs) (23)(24)26). Hepatitis E is primarily transmitted through the fecal-oral route, with an incubation period of about 15 to 60 days. The mortality rate is generally low (about 1%); however, it can reach up to 15 to 25% among infected pregnant women (7,23,24

Avian hepatitis E virus infection and possible associated clinical disease in broiler breeder flocks in Hungary

“…Vascular lesions as described for HSS in field cases (Agunos et al, 2006) or experimental studies (Sun et al, 2004b) were not observed in our field cases. It is not known how long aHEV (or associated antigen) remains present in tissues or birds, but this study suggests that infection is persistent in some birds and seropositivity also persists on a flock basis.…”

“…Histologically no neoplastic features were identified as a flock problem. Enlarged right liver lobes and subscapular haemorrhages as described in experimental aHEV (HSS) infections by Sun et al (2004b) were observed (although their inocula could also contain other agents) (Billiam et al, 2005).…”

Hepatitis E virus in humans, farm animals and animals from the sylvatic environment

“…drób domowy (kura, indyk) oraz ptaki dzikie (pustułka, kobczyk zwyczajny) (56). Przypadki HEV u kur łączone są z syndromem powiększonej wątroby (big liver syndrome, BLS), a także z zespołem zapalenia wątroby i śledziony (hepatitis splenomegaly syndrome, HSS), pomimo że wirus wykrywany był również i u zdrowych kurcząt (61). Seroprewalencja u kur wzrasta wraz z wiekiem (70).…”

Construction of an infectious cDNA clone of genotype 1 avian hepatitis E virus: characterization of its pathogenicity in broiler breeders and demonstration of its utility in studying the role of the hypervariable region in virus replication

“…Weekly serum and faecal samples were tested for avian HEV RNA by RT-PCR and weekly serum samples were tested by an avian HEV-specific ELISA with a purified recombinant avian HEV capsid antigen for anti-HEV antibodies as described previously (Sun et al, 2004). One step RT-PCR (Qiagen) and PCR premix (Invitrogen) using primer pairs specific for the Korean avian HEV strain HH-F9 were used to detect avian HEV RNA (Table S1).…”

“…At least four different genotypes of avian HEV have been identified from chickens worldwide: genotype 1 from chickens in Australia, genotype 2 from chickens in the USA, genotype 3 from chickens in Europe and China, and genotype 4 from chickens in Hungary and Taiwan (Bányai et al, 2012;Bilic et al, 2009;Haqshenas et al, 2001;Hsu & Tsai, 2014;Huang et al, 2004;Marek et al, 2010;Zhao et al, 2010). The status of avian HEV infection in chickens in Asia is largely unknown since there was only one report of avian HEV detection from China before the virus was identified recently in Korea and Taiwan (Hsu & Tsai, 2014; Kwon et al, 2012;Zhao et al, 2010).…”