Isolation, Identification And Sequence Analysis OfVP1of Duck Hepatitis A Virus Type3Strains Isolated From Shandong Province

Duck viral hepatitis (DVH) is an acute, rapidly spreading and widely distributed disease ofducklings. the mortality of the duck flock was more than90%. In the present, DHAV isbecoming a worldwide in distribution and one of the most economic importance diseases.Corresponding to the three genotypes A, B and C by neutralization tests, DHAVs had beenclassified into three serotypes: the classical serotype1(DHAV-1), a serotype recently isolatedin Taiwan (DHAV-2) and the recently described serotype isolated in South Korea and China.There is no cross-neutralization between DHAV-1and DHAV-2and limitedcross-neutralization between DHAV-1and DHAV-3. This research consists of three parts asfollows:Part1,Isolation and RT-PCR Identification of the14of DHAV-3isolatesIn this study, hepatitis samples were collected from Jinan, Weifang, Yantai of Shandongprovince from2011to2012, Epidemic materials were gringed to infect11-day-oldembryonated duck eggs and45allantoic fluid of dead embryonated eggs were harvested.The\allantoic fluid harvested were injected to five3-days-old goose of cheeper duck inmuscule with2.0ml each, The infected ducks began to dead and presented a typical clinicalsymptoms and pathological changes of duck hepatitis a virus after24hours. We designedspecific primers respectively according to the DNA sequence of Duck Hepatitis A Virse type3published in the GenBank for RT-PCR amplification, and14strains were isolated to beDHAV-3.Part2, Sequence analysis of VP1gene of duck hepatitis A virus type3isolated fromShandong province of ChinaIn this study, the viral protein1(VP1) gene of14virulent DHAV-3strains isolated fromShandong province of China in2012were amplified by RT-PCR, sequenced and analyzed.The results showed that all the VP1genes of the14strains contained720nucleotides andencoded240amino acids, with nucleotide identities of94.6%-99.9%and amino acid identities of95.0%-100%. The nucleotide and amino acid sequence homologies between the14DHAV-3isolates and other30DHAV-3reference strains were92.5%-100%and90.8%-100%, respectively. Phylogenetic analysis showed that the VP1gene of DHAV-3hadobvious geographical characteristics. All the44strains were divided into two genotypes:Chinese wild isolates belonged to genotypeⅠ (GⅠ), Vietnamese wild isolates mainlybelonged to subtype1(S1) of genotype Ⅱ (GⅡ), and Korean isolates formed subtype2(S2)of GⅡ.Part3, Molecular cloning and sequence analysis of full-length genome of SD1101strainof DHAV-3The genome sequences of duck hepatitis A virus-3（DHAV-3’SD1101strains was amplifiedby RT-PCR using primers designed according to the conserved region of DHAV-3genome.Sequence analysis showed that SD1101genetic organization was5’UTR-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3’UTR and consistented withPicornaviridae. The full genomic length of SD1101contains7774nt without Poly(A) tail,including652nt of5′UTR. Followed by a long open reading frame (ORF) of6756nt, and a386nt of3’UTR.. Comparative sequence analysis showed that SD1101strain shared93.3%-98.7%similarity at the nucleotide level and96.6%-99.2%at amino acid level withother strains of DHAV-3.