| Duck viral hepatitis, caused by Duck hepatitis virus, is an acute, rapidly spreading and widely distributed disease, which mainly infects three-week-old ducklings. In these years, the DVH caused by the Duck hepatitis A virus(DHAV-3), of which the mortality of the duck flocks was more than 80%, has been widely distributed in China and South Korea, leading to serious harm to the development of duck industry. It’s necessary to make a further research on the epidemic situation, genetic variation and molecular biological features of DHAV-3 for the object of proving the accuracy rate of the diagnosis and the effectiveness of prevention. This research consists of three parts as follows:Part 1, Sequence Analysis of Virus Isolation and Isolates P1 Gene of DHAV-3 in ShandongFrom July, 2012 to July, 2014, we got 167 samples of duck liver tissues suspected being infected by DHAV-3 collected via 18 batches of clinical investigation in four cities of Shandong including Jinan, Weifang, Yantai, Linyi. After processing the epidemic materials,vaccinating the duck embryo and artificial infection experiment, virus was successfully isolated. By using specific primers specialized in DHAV-3 testing to design a RT-PCR amplification,70 wild strains were finally isolated and identified. The results show that DHAV-3 can be detected from all the 18 batches of samples of epidemic materials, which means the detection rate was 100%; 70 in 167 samples were detected DHAV-3 positive, of which the rate reached 41.9%.From the isolated DHAV-3 samples, we selected 18 strains as representative strains for the next cloning and sequencing analysis of P1 gene. As was shown in this study, all the P1 genes of the 18 isolates, with nucleotide identities of 94.6% to 99.9% and amino acid identitiesof 95.0% to 100%, were highly congruent with another 27 DHAV-3 reference isolates.Phylogenetic analysis showed that all the Chinese wild isolates expect vaccinum isolates B63 belonged to genotypeⅠ(GⅠ), and the 18 strains identified in the study belonged to subtype 1(S1) of genotype Ⅱ(GⅡ). Among them, isolates collected from Jinan, Linyi Weifang belonged to the same subtype while Vietnamese wild isolates and Chinese vaccinum isolates B63 belonged to subtype Ⅲ of GⅡ, and Korean isolates formed subtype Ⅳ of GⅡ. The gene of DHAV-3 had obvious geographical characteristics. It was shown in the amino acid comparison analysis that there were several amino acid mutations between different subtypes,among which 22 stable ones exist. It’s 671~712 amino acids, which was important for virus antigen heteromorphosis lay in HVR. In HVR, mechanism of RGD actually mutated into QSD.Part 2, Preparation of Monoclonal Antibody of SD1101To make preparations for monoclonal antibody of DHAV-3, the BALB/c mice best immunized with the virus of the right age should have been finished with sequence analysis of full-length genome of SD1101 strain of DHAV-3 in advance. The myeloma cell lines sp2/0were fused with the spleen of immunized Balb/c while hybridomas were filtrated in an indirect ELISA. Finally, two strains of hybridoma cell lines secreting anti DHAV-3 MAb were obtained after three times of subclone, named as 4A2 and 4C3. We identified the strains obtained on the aspects of ascites titer, subtypes, amounts of chromosome, stability, specificity,and the results were as follows: Detecting serial passage through the method of indirect ELISA and using cryopreservation on alive cell supernatant, the samples detected could also secret steadily, which means no significant differences are found in its ability to secrete antibodies. The number of chromosome was about 100, revealing the typical characteristics of the chromosomes of hybridoma cell. As the hypotype identification shows, both the types of antibody, secreted by the two stains of hybridoma, were hypotype IgG1, hypotype K.Monoclonal antibody titer of ascites was 1:10240. IFA results showed that the two MAbsrespectively reacted with DHAV-3 and DHAV-1, but reacted more strongly with DHAV-3. |
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