Isolation And Identification Of DHV Sichuan Strains And Studying On VP1 Gene's Varation

In order to gain a better understanding of the prevalence of duck hepatitis virus(DHV) in China, we collected eight clinical samples which were suspected to be DHV in Sichuan. We studied on the virus isolation and indentification,the cloning and analysis of the VP1.1.Two strains were obtained through chick embryo by allantoic sacinoculation. The strains were named ZJD and ZJM respectively. The lethal rate of these isolates was 100 percent in 48h-84h when inoculating 9-day-old chicken embryos. The result showed that isolation virus strain resulted in chicken embryo stunt, hyperemic and hemorrhage and the blood spot in their liver, allantoic fluid turned green or yellow. Through recurrence test and ELD50(Embryo lethal dose) test to analyze the pathogeny of these strains. The lethal rate of 1-day-old ducklings inoculated with these isolates was 60 percent. Dead ducklings showed the sign of opisthotonos, with swollen and punctuate hemorrhage in their livers. Most of their gall bladders were engorged with bile.Some of dead ducklings are found with arborization varicose on the surface of their kidney. The ELD50 of these virus strains were 10-4.764 and 10-4.60 per 0.2ml. The test for hemagglutination was negative.2. According to duck hepatitis virus-Ⅰgenome sequence in Genebank,we designed one pair of primers in a relatively conservative region with the help of Primer Premier 5.0 and oligo 6.0 design software. Extracted the total RNA as template, we obtained 1341bp specific fragment by RT-PCR which contained VP1 gene. One out of the rest six samples was obtained for RT-PCR which was named as NX strain. Phylogenetic and evolutionary analysis of VP1 gene revealed that the homology of the nucleic acid and amino acid sequences were 91.0%～99.4％and 94.5%～99.2% between the three strains and DHV-1 reference strains. The three isolates were classified as DHVⅠ. And the homology of the nucleic acid and amino acid sequences were 93.7%～96.2% and 96.6%～97.9% among the three strains. The nucleic acid sequences comparisons indicated that the ZJD strain was close to ZZ strain by 99.4% but was distant from LY0801 by 93.1%, NX strains were near with NA by 96.6% but far from 5886 by 92.2%. ZJM strains were near with NA by 99.3% but far from 5886 by 91.0%. The amino acid sequences comparisons showed that the ZJD strain had the highest homology by 95.0% with ZZ strain,X strain,GFS99 strain while A66 strain had the lowest by 94.5%, the NX strain had the highest homology by 99.9% with NA strain while ZJ strain had the lowest by 95.0%, the ZJM strain had the highest homology by 99.2% with NA strain while ZJ strain had the lowest by 95.0%. The phylogenetic tree based on the nucleic acid sequences showned that ZJD strain located in the same branch with SG strain, ZZ strain which indicated their relationship is close. NX strain and ZJM strain were in the same branch, and both are located near with NA. The phylogenetic tree based on the amino acid sequences revealed that ZJD strain located in the same branch with 161-79-V strain. NX strain and ZJM strain were both located in the same branch with NA. Genetic relationship between nucleic acid and amino acid sequences of the three strains had some differences.In this study, three trains were obtained. the recurrence test and ELD50 test indicated these strains have a fatal on ducklings. The nucleic acid and amino acid sequences comparisons showed that the isolate VP1 gene is close to DHV-1 reference strains. The recent Sichuan strains presented lower variability.