Screening Of Specific Diagnostic Peptides Of Swine Hepatitis E Virus And Develpment Of An Indirect Enzyme-linked Immunosorbent Assay

Hepatitis E virus (HEV) is a non-A and non-B hepatitis virus. Swine hepatitis E virus (swHEV) was discovered in pigs in the USA in 1997, and it has since been demonstrated that there is potential for the zoonotic transmission and cross-species transmission between humans and pigs. Based on phylogenetic analysis, swHEV genotypes 3 and 4 contain genomic sequences closely related to human HEV. Therefore, there is a real need for specific and effective methods of prophylaxis, diagnosis and treatment for this disease.The antigenic structure of swHEV is yet to be fully investigated. The research on swHEV has been limited mainly to local epidemiological studies and simple clinical analysis of animals. The commercially available kits to detect swHEV are the same as the kits used to detect human HEV. Despite the fact that there is high homology between the genomic sequences of swHEV and human HEV, there are still important differences and minor changes which could affect the sensitivity and specificity of the diagnostic test when applied to animals. Therefore, the results which used a human HEV epitope to detect swHEV were not very satisfactorily. So, by bioinformatics analysis, we identified and then synthesized 12 peptides from open reading frames (ORFs) ORF1, ORF2 and ORF3, including swHEV1-swHEV12. Using the results from ELISA, we selected swHEV11 as the best candidate antigen and used it as a coating antigen for the development of peptide-based swine anti-HEV ELISA kits. The study of the swHEV11 epitope might not only provide an efficient way to develop a kit which detect swHEV rapidly and accurately, but might also be applied to the development of vaccines for swHEV to control the spread of HEV and the prevention of this virus passing from pigs to humans. By computer software DNAStar(Madison,Wisconsin,USA)analysis, we identified and then synthesized 12 peptides from open reading frames (ORFs) ORF1, ORF2 and ORF3, including swHEV1-swHEV12 from Xinjiang strain(AY594199). Using the results from ELISA, we selected swHEV-11 as the best candidate antigen and used it as a coating antigen for the development of peptide-based swine anti-HEV ELISA kits. The synthetic HEV peptides were further purified and used the prepared HPLC to detect the purity of synthetic peptides. These 12 peptides were coated on microtiter plates. We detected the immune response of IgG activity by indirect ELISA test. The same rate of positive sera was 87.5% (21/24), and the same rate of negative serum was 100.0% (6 / 6). Our findings strongly suggest that peptide swHEV-11 is a potent diagnostic reagent of swHEV that could be used in the development of highly efficient diagnostic assays for the specific and highly sensitive detection of anti-HEV activity in swine serum samples.An indirect ELISA assay using synthetic peptide from one sequence of the open reading frame (ORF) 3 of swine HEV was developed for the detection of swine hepatitis E virus. This assay was optimized for an antigen coating concentration of 3μg/mL and a serum dilution of 1:10, with a standard incubation time of 30 min. The optimal dilution and reaction time of enzyme labeled antibody was 1:12000 and 60 min. Swine HEV ELISA kit was developed for detecting anti-HEV IgG by using synthetic peptide swHEV11. When the kit was applied to detect HEV antibodies in sera of pigs, the results were quite consistent with the HEV diagnostic kit from Wantai. The conformity rate reached 73.4% (205/279), and the sensitivity and the specificity reached 73.3% (200/273) and 83.3% (5/6), respectively. The coefficient of variation (CV)<10% in the same batch, and <20% in different batches. This is the first systemic study to screen the diagnostic peptides of swHEV and our findings strongly suggest that peptide swHEV11 is a potent diagnostic antigen of swHEV, which could be used in the development of highly efficient diagnostic kits for the specific and highly sensitive detection of anti-HEV activity in swine serum samples.In summary, our results indicated that swine HEV ORF (ORF1, ORF2, ORF3) has epitope swHEV11, which located at position 91-114 of the HEV ORF3. This is the first systematic Screening diagnosis of swine HEV polypeptide. Our results showed that swine HEV polypeptide swHEV11 was an effective diagnostic reagents, and can be used for the development of efficient diagnostic kits to be specifically and highly sensitive detection of HEV antibody activity in serum samples. The results indicated that the application of indirect ELISA and the HEV ELISA kit is specific, sensitive and stable, may provide a simple and rapid method for monitoring serum antibodies against HEV infection and seroepidemiological surveys of HEV infection.