Isolation And Identification Of Duck Hepatitis A Virus And The Development Of An ELISA For Detectng Egg Yolk Antibodies Against DHAV-1

In recent years, duck hepatitis virus (DHV) is still an epidemic disease in many provinces including Guangxi in china. Duck hepatitis A virus (DHAV) is the causative agent of duck viral hepatitis and has three different serotypes. Therefore, it is necessary to isolate and identify the duck hepatitis virus circulating in Guangxi in resent years and establish an indirect ELISA method for detecting the antibody's level of the vaccined breeding ducks to provide useful material and technique for study of the etiology and detection of antibody in the future. It is of great significance for epidemiological studies, prevention and control of the disease.In this study, twelve clinical samples were collected from suspected for DVH and they were processed and used to isolate and identify the DHAV with the conventional technique. Then the isolates were typed by the serological neutralization test and a duplex real-time RT-PCR assay for the identification of DHAV-1 and DHAV-3. The results showed that seven isolates were recovered from the clinical samples and named as GXNN01, GXNN02, GXGL01, GXLZ01, GXLZ02, GXNN03, and GXGL02, respectively. The virus isolation rate was 58.33%.The Isolates GXNN01, GXNN02, GXGL01, GXLZ01, GXLZ02 were clustered in the DHAV-1 serotype and GXNN03, GXGL02 were clustered in the DHAV-3. The results of serotyping are consistented with that of duplex RT-PCR. Sequencing and analysis of the nucleotide sequences of the main structure protein VPl were also done with conventional molecular biological technology. The results indicated that the nucleotide and amino acid identities between five DHAV-1 isolates were 91.0%-98.0% and 93.3%-98.7% respectively. They are 91.0%-98.0% and 91.2%-99.2% compared with that of other DHAV-1 representative strains. The nucleotide and amino acid homology of VP1 protein genes of the two DHAV-3 isolates were 95.4% and 95.9% respectively. And they were 90.4%-98.3% and 91.3%-98.3% compared with that of other used DHAV-3 representative strains. All the isolated virus are less than 20% compared with that of DHAV-2 representative virues.According to the fact that DHAV-1 is the main epidemic genotype of the DHAV in Guangxi, the differential and ultracentrifugation were used to prepare DHAV-1 antigen, and the egg yolk antibody was extracted and purified by the salting-out method. An indirect ELISA for detecting egg yolk antibodies against DHAV-1 has been developped and the reacting conditions were optimized. The results demonstrated that the optimun conditions of the method were as following:The concentration of viral antigen for coating plate was determined to be 6.78 ?g/mL, of secondary antibody(anti-duck IgG/HRP antibody produced in rabbit) was 0.31?g/mL, of sample antibody was 10.37 ?g/mL, of the sensitive measurement value was 0.08?g/mL. The OD450 critical value was 0.233. The method was specific and repeatable. The established method was used to detect 40 serum samples and 80% samples was positive. So the method is simple, intuitive, and suitable for the detect DHAV-1 egg yolk antibody in clinic.This study was isolated to 7 DHAV viruses, including 5 DHAV-1 and 2 DHAV-3, analyze their genetic evolution of the major structural protein gene VP1, the results showed that the homology was low of the DHAV-2 virues. At the same time successfully established detection ELISA method of DHAV-1 yolk antibody. The results of the research will provide useful materials and technical support for the research of DHAV epidemiology in Guangxi and the immunization level of the duck DHAV-1 and the disease control and prevention.