| Recently, duck hepatitis A virus genotype C(DHAV-C),caused duck viral hepatitis, hasbeen widespread in China and South korea which becomes a challenge to duck industry. Theepidemiology, clinical symptoms and pathogenesis of DHAV-C are very identical to those ofDHAV-A, making its difficult to distinguish DVH. Therefore, the study on rapid diagnosis ofDHAV-C has great significance. In this research, the isolation and identification of DHVpopular in Sichuan were done and two assays were developed: a real-time PCR for detectingduck hepatitis A virus genotype C (DHAV-C)which was applied in the research of dynamicdistribution of the virus in duck, and a duplex RT-PCR assay for detecting duck hepatitis Avirus genotype A (DHAV-A) and genotype C. The main achievements were as followed:1. Duck hepatitis A virus genotype C was confirmed to be the main cause of duckhepatitis in Sichuan province.In this study,24clinical liver samples collected from ill ducks suspectedduck viral hepatitis, which characterized by enlargement, bleeding lesions from10duck farmsin Sichuan province from November2009to July2011were used for virus isolation. Total16strains of virus were obtained and identified as Duck hepatitis A virus genotype C by RT-RCR.Furthermore,4clinical isolates selected randomly caused same symptoms and pathologicalchanges as clinical cases in ducking infected those virus This results confirmed that Duckhepatitis A virus genotype C is the main cause of Duck viral hepatitis currently in Sichuanprovince.2A duplex RT-PCR assay for Simultaneous detecting duck hepatitis A virus genotypeA(DHAV-A) and genotype C (DHAV-C) was established successfully.The epidemiological characteristics, clinical symptoms and pathological changes ofDHAV-C were very similar to those of DHAV-A, making laboratory testing to be importantfor distinguishing these two virus. According to complete genome sequences of DHAV-A andDHAV-C from GenBank, two pairs of primers were designed based on the genomicsequences of3C,3D gene of DHAV-A and2C gene of DHAV-C. After the optimization ofreaction conditions and the validation of specificity, sensitivity and repetitiveness of themethod, a duplex RT-PCR assay was developed. The assay was confirmed to be specific andsensitivity of the assay was4.98×104copied/μL（6.3fg） for DHAV-A DNA and1.68×104 copies/μL（3.4fg）for DHAV-C DNA respectively.The clinical samples collected from April2007to July2011were detected by the duplex RT-PCR. The positive rates of DHAV-A andDHAV-C were20%（14/70）,70%（49/70）, respectively, Randomly16positive samples ofDHAV-C tested by the duplex RT-PCR was further Conformed by virus isolation. Theresults show the duplex RT-PCR assay developed this study is a sensitive and specific methodfor clinical differential diagnosis of the infection of duck hepatitis A virus genotype A orgenotype C, and can be used for the detection of clinical tissue samples directly.3A real-time PCR method for detecting duck genotype C hepatitis virus was establishedinitially.Until now, there is no report about real-time quantitative PCR detection of DHAV-C. Inthis study, a pair of primers were designed targeted on the genomic sequences of2C ofgenotype C hepatitis virus and a real-time quantitative PCR method for detecting thegenotype C hepatitis virus of ducks was established successfully.The assay was confirmed tobe specific and stability. The inter-assay coefficient and intra-coefficient of variation wererange from1.02%to1.77%and0.15%to2.0%; the amplification efficiency was97.9%;linear relationship was from1.68×104to1.68×109copies/μL;the minimum detection limitwas1.68×103copies per μL;the melting curve analysis showed a single specific peak andthere were net non-specific amplification and primer dimers.38clinical samples suspectedduck hepatitis were detected by this assay,the result showed that22samples werepositive,and other16samples negative to genotype C hepatitis virus were further indentifiedas genotype A hepatitis virus, this result was further confirmed by virus isolation. Collectively,the real-time PCR is specific and sensitive, and is suitable for rapid detection of clinicalsamples for the diagnosis of duck genotype C hepatitis virus4The dynamic quantitative distribution of genotype C hepatitis virus in ducks has beencompleted.The dynamic quantitative analysis of genotype C hepatitis virus in SPF ducks aremeasured by the real-time PCR at1h,6h,12h,18h,24h,48h and72h after infection. Theresults revealed that the DHAV-C could be detected earliest in the liver during12hour postinfection. Moreover, the high viral loads were identified in the heart, liver, spleen, lung,kidney, bursa of Fabricius, thymus, pancreas, brain and small intestine at24hour postinfection. Collectively, this study provided a valuable data basis for the research of pathogenicity of DHAV. |
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