Avian hepatitis E virus infection of duck, goose, and rabbit in northwest China

To the Editor: Hepatitis E virus (HEV) belongs to the Hepeviridae family, which consists of two genera, Orthohepevirus and Piscihepevirus. Within the Orthohepevirus genus, four viral species, designated A–D, infect a wide range of mammalian species, including human, swine, wild boar, chicken, rat, ferret, rabbit, mongoose, camel, cow, and bat. Within the species Orthohepevirus A, various HEV sequences are grouped into eight genotypes. Genotypes 1 and 2 exclusively infect humans, whereas genotypes 3 and 4 infect humans and numerous other animal species. Genotypes 5 and 6 have only been isolated from wild boar, whereas genotypes 7 and 8 have been isolated from camels. Recently, it was recognized that strains of HEV genotypes 3 and 4 can cross-species barriers and are thus zoonotic genotypes. In addition, it has been documented that HEV genotype 7 isolated from camels may also infect humans. However, Orthohepevirus A genotypes 5, 6, and 8, as well as various HEV strains of Orthohepevirus C and D, have not yet been reported to cause zoonotic infections. Avian HEV, a member of Orthohepevirus B, has been isolated from chickens with big liver and spleen disease, also known as hepatitis-splenomegaly syndrome . The avian HEV genome shares ~48% identity with mammalian HEVs. In several previous studies, under experimental conditions, avian HEV could infect turkeys, but not rhesus monkeys or pigs. To investigate the prevalence of avian HEV infection in the northwest region of China, we collected sera, fecal swabs, and bile samples from a mixed group of animals consisting of 57 chickens, 30 ducks, 24 geese, and 16 rabbits cohabitating within a shared space in March 2017. Detection of antiviral serum IgG antibodies is often used as evidence of prior infection. We detected serum anti-avian HEV antibodies in four animal species by indirect ELISA using truncated avian HEV ORF2 protein as the coating antigen and a blocking ELISA protocol described by Liu et al. Based on the cutoff value of this method, the results showed that 20/57 chickens, 9/30 ducks, 6/24 geese, and 8/ 16 rabbits were positive for anti-avian HEV antibodies (Supplementary Figure S1). Because the results demonstrated the presence of specific anti-avian HEV antibodies in all species tested, avian HEV appears to infect chickens, ducks, geese, and rabbits within a mixed animal group. As further confirmation of avian HEV infection of these four species, fecal swabs and bile samples were collected from the animals to test for avian HEV ORF1 and ORF2 RNA. First, total RNA was extracted from 100 μL of bile or from 10% suspensions of fecal swab samples using TRIzol Reagent (Invitrogen, Burlington, Ontario, Canada) followed by RT-nPCR. The two specific primer pairs used for RT-nPCR detection of avian HEV ORF1 and ORF2 RNA are described by Dong et al. and Sun et al., respectively. Fecal swab analysis detected 11/57 chickens, 8/30 ducks, 2/24 geese, and 2/16 rabbits positive for ORF1 (Supplementary Table S1). Additionally, we found 32/57 chickens, 12/30 ducks, 3/24 geese, and 6/16 rabbits positive for ORF2. From the bile samples, we identified 1/ 4 chickens, 1/4 ducks, 1/4 geese, and 1/4 rabbits positive for ORF1 (Supplementary Table S1). However, 2/4 chickens, 2/4 ducks, 1/4 geese, and 3/4 rabbits were positive using the ORF2 primers (Supplementary Table S1). Notably, the results showed that all positive samples for the ORF1 gene were also positive for the ORF2 gene. One possible reason for the increased detection of virus based on ORF2 in both fecal and bile samples