| Duck viral hepatitis (DVH) is known as an acute, highly lethal viral disease within 3 weeks old ducklings by duck hepatitis virus, seriously affecting the development of duck industry. At present, the main pathogen of DHV is the duck hepatitis A virus (DHAV) in China. Therefore, it is significant to conduct studies of DHAV isolation, genetic variation, main structural protein expression and differential diagnosis of pathogen for future research on DHAV molecular epidemiology, pathogen detection and disease prevention and control.A strain of virus, named as LS strain, was isolated and identified by duck embryo proliferation, animal regression test, ELD50 and RT-PCR method from 1 case of suspected DVH. The complete genome sequences of the isolate were amplified, cloned and analyzed. The result showed that the whole genome of LS strain was 7 815 nt in length excluding the cap structure, including 5’UTR (652 nt), ORF (6 756nt) and 3’UTR (366nt) back with at least 26A poly (A) tail LS strain shared the highest nucleotide and amino acid homology with DHAV-3 reference strains reaching 97.4% and 99%; furthermore, LS strain and DHAV-3 C-BLZ, SD1201 strain were in the same genotype, and they might come from the same ancestor. As a result LS strain could be ranked DHAV-3 style.Amino acid sequence and the major epitopes of LS strain VP1 structural protein was done by relevant molecular biology techniques and the highly variable region was expressed by prokaryotic expression system. The results showed that the 132-139,178-191,195-203,211-221 amino acid segments are likely to become B cell epitopes. The 132-240 amino acid segment of the hypervariable region was successfully expressed with the molecular weight of the purified recombinant fusion protein about 38ku. Western-blot result showed that the fusion protein can be identified by anti GST tag monoclonal antibody with a good reactogenicity.This study also successfully established a DHAV FQ-PCR genotyping method based on melting curve with SYBR Green I fluorescent dye and a pair of specific primers designed according to the 1,3 types of duck hepatitis virus 5 conserved region. The results show that the established method was specific, rapid and sensitive. The method had a wide linear range of 1.0×102 to 1.0×109 copies/μL concentration template for detection. The detection limit of this assay was 100copies/μL,100 times compared with conventional PCR. The method has a good specificity excluding the common fowl source virus. The detections of high, medium, low different concentrations of plasmid standard show good repeatability. The study of proliferation in tissues of ducklings infecting with DHAV-1 and DHAV-3 with the established method were also performed. The results indicated that the infection of DHAV exists extensively in tissues. The DHAV-1 and DHAV-3 strain used in this study had the same characteristics of replication in tissues. The virus titer of the liver, spleen, kidney and lung was very high and the virus titer of liver was highest. Therefore, the results explained the characteristics of lesions caused by DHAV infection of duck in the main target organs such as liver, spleen, kidney and so on. |
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