Analysis of Avian Hepatitis E Virus from Chickens, China

Avian hepatitis E virus (HEV) has been identified in chickens; however, only 4 complete or near-complete genomic sequences have been reported. We found that the near-complete genomic sequence of avian HEV in chickens from China shared the highest identity (98.3%) with avian HEV from Europe and belonged to avian HEV genotype 3.


The Study
In May 2009, hepatitis-splenomegaly syndrome affected a fl ock of 37-week-old broiler breeder hens in Shandong, China. This fl ock had a history of decreased egg production. Affected chickens had regressive ovaries, extensive necrosis and hemorrhage of the liver, and enlarged liver and spleen. Antibodies against avian HEV ORF2 were detected in 80 of 94 serum samples from the same chicken fl ock, according to ELISA (5,10) with the truncated ORF2 protein used by Guo et al (10) and chicken serum diluted 1:100 in 0.5% Tween-20 phosphate-buffered saline containing 2.5% nonfat dry milk and 10% Escherichia coli lysate. On the basis of previous results, we used a cutoff optical density of 0.43 (11). Using a published method (12), we detected an avian HEV ORF2 RNA gene with 242 bp in 7 of 10 fecal and 5 of 8 bile samples.
From the bile samples that were positive for the avian HEV ORF2 gene, we used nested RT-PCR with 5 overlapping fragments to amplify the near-complete genomic sequence of avian HEV. Primers were designed on the basis of the other 4 avian HEV near-complete sequences in GenBank ( Table 1). The RT-PCR conditions and reaction mixture were designed according to the SuperScript II One- Step RT-PCR System instructions (Invitrogen, Carlsbad, CA, USA). To identify the extreme 3′ genomic sequence, we used a modifi ed RACE (3′ rapid amplifi cation of cDNA ends) technique. The sense primer F5 (Table 1) was chosen from the ORF2 region, and the antisense primers included a commercially available anchored adaptor primer and an amplifi cation primer (Invitrogen). Using inner PCR prim- ers, we sequenced the PCR products of 5 fragments in both directions (Table 1); the sequence data were collected by an ABI3730 Genetic Analyzer (JinSiTe Biotech Co., Nanjing, China). We assembled the near-complete genome of avian HEV, which was 6,660 nt long including the 3′ poly A tail, by using 5 overlapping fragments sequences and Lasergene 7.0 EditSeq computer programs (DNAStar, Madison, WI, USA) and designated it China avian HEV (CaHEV). CaHEV contained a complete ORF1 gene encoding a nonstructural protein of 1,522 aa, an ORF2 gene encoding a capsid protein of 606 aa, an ORF3 gene encoding a cytoskeleton-associated phosphoprotein of 87 aa, and a 3′ noncoding region of 121 nt. The sequences of CaHEV were deposited into GenBank under accession no. GU954430.
ORF1 of CaHEV contained most mutations compared with prototype avian HEV (prototype aHEV); 5, 16, and 29 nonsilent mutations occurred in the methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp) functional domains, respectively (data not shown). However, only 2 mutations occurred in motif VII of RdRp domain (Figure 1, panel A), which contains 8 motifs responsible for virus replication (13). The 2 mutations in motif VII of the CaHEV RdRp domain are L(1432)M and I(1434)V. Australian avian HEV isolate (AaHEV) also has the mutation in the latter position and was a transition from I(1433) to T (Figure 1, panel A). This position is well conserved among mammalian HEV isolates by the presence of V, which is the same as CaHEV (Figure 1, panel A).  In the ORF2 region, 6 nonsilent mutations (C4R, R5G, G27S, T42A, T303V, and Q473M) were determined for CaHEV and compared with prototype aHEV. One mutation of Q(473)M, in the antigenic domain II, was seen in EaHEV and in CaHEV (Figure 1, panel B). Because this domain is unique to avian HEV, as predicted by Haqshenas et al. (14) and Guo et al. (10), this point mutation may change the antigenicity of the epitopes in domain II of the capsid protein. In antigenic domain IV, a mutation of R(600)K occurred in the "avirulent aHEV" compared with other 4 avian HEV strains, including CaHEV, from the sick chickens ( Figure 1, panel C). This mutation may affect the virulence of avian HEV as speculated by Billam et al. (8) and Billic et al. (9). The 3 putative N-linked glycosylation sites ( 255 NLS [1], 510 NST [2], and 522 NGS [3]) are shared between prototype aHEV and CaHEV (data not shown). However, the second site is 510 NNT in "avirulent aHEV" and AaHEV strains and is eliminated in the EaHEV strain (9). In human and swine HEV strains, these sites are 137 NLS (1), 310 NLT (2), and 562 NLS (3). Recently, the potential Nlinked glycosylation in ORF2 was shown to prevent formation of infectious particles, but its role in other functions of HEV, e.g., virus virulence and cell tropism, remain to be elucidated (15). In the ORF3 gene, including only 83 aa, 10 nonsilent mutations were found compared with the prototype aHEV, and 9 mutations were the same as EaHEV (data not shown).
Phylogenetic trees of the near full-length sequence of avian and mammalian HEV strains were constructed by using the neighbor-joining distance method and Lasergene  7.0 software. A bootstrap test of 1,000 replicates was used to evaluate the reliability of the groups. Avian HEV was segregated into a distinct branch separate from mammalian HEV; according to the genotype separation corresponding to their geographic origin suggested by Bilic et al. (9), Ca-HEV belongs to avian HEV genotype 3 ( Figure 2).

Conclusions
Avian HEV infection of a chicken fl ock in Shandong, China, was identifi ed by detection of avian HEV ORF2 antibodies and viral RNA. A near-complete avian HEV genome from the fl ock was determined, and sequence analysis indicated that this avian HEV strain displayed the highest identity (98.3%) with EaHEV and belonged to avian HEV genotype 3.
This study was partially funded by the Taishan Scholar project of Shandong Province.
Mr Qin Zhao is a PhD student in the laboratory of "Taishan Scholar" Immunobiology at the College of Veterinary Medicine, Shandong Agricultural University. His research interests are avian HEV pathogenesis and immunology.