Isolation And Identification Of Duck Hepatitis Virus And The Development Of A RT-PCR Detection Technique

Duck viral hepatitis (DHV) is a severe hepatitis which is caused by duck hepatitis virus (DHV), and results in a severe loss by a highly morbidity and mortality in ducklings. Recently there is an emerging of hepatitis in the field which can not be prevented and controlled by the conventional type I vaccine in some provinces in China. It is important and necessary to isolate and identify the field strains of DHVs and develop a rapid diagnosis technique for the effective control of the disease.16 DHV strains were isolated by inoculating allantoic sac of chicken embryos from 31 clinical samples which is suspected to be DVHs. The neutralization tests were done in chicken embryos with the serum to DHV type I (DHV -I). Isolates 050028, NN4, HP, 050058, 050044, ZHJB, NN3 can be neutralized, isolate 040146 can be partially neutralized, isolate ZHJA can not be neutralized at all. At the same time, the reference strain SHDE5 can not be neutralized by the anti-ZHJA serum since isolate 040146 can be partially neutralized. It meant that isolates 050028, NN4, HP, 050058, 050044, ZHJB, NN3 belong to DHV-I, isolate040146 belongs to DHV-I variant (Iv DHV), isolate ZHJA belongs to a new serotype of DHV. And the rest 7 isolates can't typing since their CEID50s were too low.In this study, three oligous primers (DHVA1, DHVA2, DHVTs) which is set in VP0 gene of DHV, were designed to run a primary reverse transcription polymerase chain reaction (RT-PCR) and heminested PCR. Both of these assays can amplify all of reference strains and 16 field isolates. 25 clinic samples, which were collected from different places of Guangxi, were detected by using developed primary RT-PCR and heminested PCR, and 12 (48%) of them were positive in the primary RT-PCR and 21 (84%) of them were positive in the heminested PCR and obtained the bands of 426bp and 293bp respectively. The heminested PCR can significantly increase the sensitivity of the detection.A partial fragment of VPO and 3D gene of some isolates were amplified by RT-PCR and sequenced respectively, then compared with the sequences of reference viruses. The nucleotide sequences comparisons showed that there were 90.3%～99.5% homology between 6 isolates on VPO gene, and the isolate HP had the highest homology by 99.7% with vaccine strains while isolate ZHJA had the lowest by 91.1%. The phylogenetic tree base on the sequences of these genes shown that isolate HP located in the same branch with the vaccine strains used in China and other isolates set in another branch, it is identical to the results of neutralization test. On 3D gene, the reference strain SHDE5 located in the same branch with E53 while other isolates set in another branch. There is no evidence show that 3D is related with the serotype.The results of this study demonstrated that the recently prevalent DHVs in Guangxi are mostly belonged to type I, and there is also type Iv and new serotype exist. The neutralization test is a reliable method for identification and serotyping for the new isolate, and RT-PCR technique is a rapid, sensitive and specific for detecting the DHV from the clinical samples directly. The results of sequence analysis indicated that the VPO gene may represent the serotype of DHV, and no evidence show that 3D is related with the serotype.