Isolation, Identification And Antigenicity Of Avian HEV From China

Avian hepatitis E virus (HEV), the causative agent of big liver and spleen (BLS) disease or hepatitis-splenomegaly (HS) syndrome, mainly infects 30-70 week old aged broiler breeder hens and laying hens and causes increasing mortality, egg reduction (20-40%), red fluid in the abdomen, regressive ovaries and enlarged liver and spleen in chickens. The diseases caused heavy economic losses of Australia poultry in 1980s. There were also few case reports in China.Avian HEV, which belonged to the Hepeviridae family, was first isolated and characterized from chickens with HS syndrome in USA. Compared with mammalian HEV complete genomic sequences, avian HEV showed only 50% identities with mammalian HEV. But avian HEV is antigenically and genetically related with swine and human HEV. The full length of avian HEV genome is 6.6kb and is 600bp shorter than the length of mammalian HEV complete genome. The complete genome of avian HEV contains three open reading frames (ORF), ORF1, 2 and 3, besides the 5'cap and 3'PolyA structure. The ORF2 gene encodes the capsid protein and contains primary epitopes of avian HEV. Because there was no effective cell culture system for avian HEV, many researches focused on the ORF2 protein of virus substructure protein. Recently, the ORF2 protein of avian HEV expressed with prokaryotic and eukaryotic expression system has been characterized and was used to diagnosis for antibody test.1. Detection of avian HEV from China with RT-PCR method.Fecal, bile and serum samples were collected from chickens with HS syndrome or BLS disease in some fields, that there were high positive rate for avian HEV antibody according to the previous results of seroepidemiology investigated in our lab. Partial ORF2 gene of avian HEV was detected using RT-PCR method based on the primers described by other researchers. Partial ORF2 gene sequences was obtained from a flock of 35 or 37-week-old broiler breeder hens with HS syndrome. Meanwhile, sequences analysis revealed that the obtained sequences showed the highest identity with European isolates and phylogenetic trees showed that the isolates in this study are closely related to the Europe isolates and belonged to avian HEV genotype 3. 2. Analysis of the complete genome of avian HEV from China.In the study, twelve primers pairs were designed according to the referenced sequences in GenBank. Then six overlapping fragments were amplifying from the bile samples to be positive for avian HEV RNA. The complete genome of Chinese avian HEV isolate was assembled by using the 6 overlapping fragments and designated CaHEV. The complete genomic and different region sequence analyses indicated that CaHEV shared the highest identity (98.3%) with European avian HEV isolate. Compared the ORF2 amino acid sequences with avian HEV each other, the ORF2 gene shared 98.3%–99.7% aa sequence identities with each other. It indicated that there was one serotype among avian HEV isolates. ORF1 of CaHEV contained most mutations compared with prototype avian HEV. Phylogenetic trees of the full-length sequence of avian and mammalian HEV strains showed that avian HEV was segregated into a distinct branch separate from mammalian HEV and CaHEV belongs to avian HEV genotype 3.3. Pathogenesis research of avian HEV from China.Avian HEV stock of bile and fecal samples suspension were collected from challenged chickens inoculated with bile samples positive for avian HEV RNA detected with RT-PCR. Then the avian HEV stock was filtered and quantitated using GE method. In the study, eighty-four chickens of 15 week ages were divided into 3 groups which were separately housed. Chickens in two groups were inoculated with CaHEV stock by intravenously and orally, respectively. The third group was control group. Fecal samples were collected from the challenged chickens every one day post inoculation and serum samples were collected from every three days post inoculation. Meanwhile, two chickens were necropsied every one week post inoculation and the remaining chickens were necropsied in 12 wpi. Bile and live, spleen tissues were collected. The detection of all collected samples for avian HEV RNA using RT-PCR indicated that avian HEV RNA was first detected from fecal samples of challenged chickens in 3 dpi and was still detected in 54 dpi in intravenously group. Viremia in this group was first detected in 6 dpi and persisted to 21 dpi. In orally group, avian HEV RNA in fecal samples was first detected in 6 dpi and still in 62 dpi and viremia was first appeared in 12 dpi and disappeared in 33 dpi. Analysis of seroconversion to avian HEV antibody using indirect ELISA showed that the antibody titers of intravenously group wane rapidly when it reached the peak, but the antibody titers of orally group declined slowly when reached peak. In addition, partial chickens showed big live and spleen and hemorrhage of the liver and spleen in the two challenged group. Meanwhile, lymphocytic periphlebitis and phlebitis foci were observed in the liver sections from two challenged group. These results further confirmed the relationship between avian HEV and HS syndrome.4. Development and application of indirect ELISA for the detection of avian HEV antibody.An indirect ELISA was established for the detection of avian HEV antibody using the coated antigen of avian HEV truncated ORF2 protein through optimizing the different conditions. We investigated epidemiology of 11 chicken farms in Shandong province for avian HEV infection using the indirect ELISA. It was shown that avian HEV antibodies were detected in all 11 flocks. But the positive rate for avian HEV antibody among 11 flocks was significant difference. The positive rate reached 77% in two flocks. There were 493 of positive serum for avian HEV antibody in 1379 serum collected from the 11 flocks. It was suggested that avian HEV infection has been very prevalence in Shandong province.In addition, we also detected the serum against antibody from different aged chickens in five flocks of same chicken farm using the indirect ELISA. It was shown that the serum from 12 week-old-age chickens were positive for avian HEV antibody and the positive rate reached the largest in the serum from 20-30 week-old-aged chickens in all five flocks. In a flock, the positive rate reached the largest again in the serum of 40-50 week-old-aged chickens. Only 10% of chickens older than 60 weeks were seropositive. These results suggested that the chickens from five flocks were naturally infected by avian HEV in younger than 10 weeks according to the results of the serum from 11 or 12 weeks to be positive and the antibodies to avian HEV of mostly chickens older than 60 weeks disappeared.5. Seroconversion to avian HEV antibodies against different protein in inoculated chickens.In previous study, it was documented that primary epitopes located in the capsid protein of avian HEV and six antigen domains located in the 22 to 83aa, 339-383aa, 389-410aa, 461-492aa, 556-566aa and 583-600aa, respectively. In addition, ORF3 protein of avian HEV can induce immune responses in chickens. Then to investigate the difference of seroconversion to avian HEV antibodies against the different domains located in ORF2 protein and ORF3 protein, the serum from challenged chickens of different dpi were detected with indirect ELISA using different coated antigen including ORF2-F (22-83aa),ORF2-S(339-606aa),ORF2-5(339-442aa),ORF2-8(383-515aa),ORF2-9(412-546aa)and ORF3 protein. The results showed that the antibodies against ORF2-S were first detected in the serum from challenged chickens of 12 dpi and antibodies against ORF2-F and ORF3 was first detected in 15 dpi serum. But the titers of antibodies against ORF2-F and ORF3 wane rapidly and the titers of ORF2-S declined slowly when they reached the peak, respectively. It was suggested that antibodies responses to the antigen domain of 22 to 83aa located in the forward fragment of ORF2 protein and to ORF3 protein were transient and antibody responses to the latter fragment of ORF2 protein. The titers of antibody against ORF2-5 (339-442aa) in the serum of different dpi were similar as the titers of ORF2-S (339-606aa) detected with indirect ELISA. It was suggested the antigen domains located in 339 to 442aa primarily induced immune responses in the challenged chickens. In addition, according to the titers of antibody against ORF2-8 (383-515aa), we concluded that the 339 to 383aa region was the primary antigen domain in the latter fragment of ORF2 protein induced immune responses in inoculated chickens. Meanwhile, there were no antibody responses to the 412 to 546aa region of ORF2 protein in inoculated chickens because of no reaction between serums of different dpi with ORF2 protein using indirect ELISA.6. Self-assembly of empty virus-like particles of avian HEVThree truncated proteins including complete ORF2 protein, deleted 56 amino acids in N terminal (ORF2Δ56) and C-terminal 268 amino acids (ORF2-268) were expressed with baculovirus system. Three truncated ORF2 proteins were successfully expressed in baculovirus system through identification of IFA and Western-botting methods. Meanwhile, it was found that the ORF2Δ56 protein was secreted into the cell culture medium when it was expressed with baculovirus system in sf9 cells. Compared with the proteins of ORF2 and ORF2Δ56 expressed with E.coil system, it was shown some proteins to be size smaller than expected protein were produced in baculovirus system. The reason is that the protein expressed with baculovirus may be proteolytic processing in the sf9 cells. The empty virus-like particles of avian HEV was examined with EM when the protein secreted in cell culture medium expressed with baculovirus for ORF2Δ56 protein was purified.