Isolation And Identification Of A Duck Parvovirus And Sequence Analysis Of Ns Gene And Sequencing Of The Genome Of An H9N2 Avian Influenza Virus

The duck parvovirus disease is an acute and contagious disease caused by the Duck Parvovirus(DPV). It is one of the serious diseases which block the development of duck raising by causing the death at 50~80% . Avian Influenza Virus (AIV) is the member of Influenza virus A of Orthomyxoviridae which causes the avian infection and disease. H9N2 subtype AIV has not high pathogenicity but it spreads widely. H9N2 subtype AIV can collaborate with other pathogen and make reservoirs immunosuppression. The hazad of DPV and AIV to poultry raising cannot be neglected.In this research, as regard of DPV, some dead ducks' liver tissues were sample from several duck farms which broke out the duck parvovirus disease in the region of Yuyao of Zhejiang Province in April, 2004. From the suspension of the dead ducks' liver tissues viruses partickles was isolated and after the purification of viruses by the sucrose density gradient centrifugation, a typical DPV particle was observed as the size of 20nm by electron microscopy.In the test of double agar-gel immunodiffusion, a line of precipitation between the anti-DPV serum and the pathogen was visible. Through SDS-PAGE, three main segments of structural proteins were observed which were consistent with the reported segments of the standard Duck Parvovirus.With a pair of primers designed according to the sequence from 979bp-1566bp of the gene of ns of DPV in GenBank, a fragment was amplified by PCR as expectation. The amplified DNA was cloned into pMD18-T vector and sequenced. Sequence indicated that both DNAs of the amplified and the DPV sequence in GenBank displayed 98% identity.The result of the animal test indicated that the mortal rate is 75%. The syndrome of the sick young duck and the symptom of anatomical pathology were obvious. The infection of duck hepatitis virus type I, avian influenza virus or Newcastle disease virus was excluded by the antiserum reaction.The research of H9N2 subtype AIV chose a virus ,Ck/HB/03, isolated from chickens in 2003 as a subject. Eight full-length genes of Ck/HB/03, HA, NA, M, NS, PA, PB1, PB2, NP, were amplified by RT-PCR. Then the eight full-genes were cloned respectively into the vector, pMD-18T and sequenced.After analyzing the homology of the nucleotide sequences of Eight full-length genes of Ck/HB/03 by Clustal X and DNAStar with the viruses in Genbank, the results indicated that the homology between the NA Gene nucleotide sequence of Ck/HB/03 and the isolated H9N2 subtype AIV from Hong Kong (Ck/HK/99) was 98.2% . The other 7 genes of Ck/HB/03 displayed high homology with isolated viruses from China. The homology of deduced amino acids sequences of HA and NA was 86.6%~ 98.0% % 88.7%~99.4%.Phylogenetic trees are generated by Phylip3.5. A/turkey/Wisconsin/66(H9N2) is used as out-group. The results of phylogenetic analysis showed that the NA Gene nucleotide sequence of Ck/HB/03 and Ck/HK/99 shared the same branch. The other 7 genes of Ck/HB/03, Ck/BJ/98, Ck/HN/98 and Ck/NX/99 shared one branch and belonged to the same sublineage with other Chinese isolated H9N2 subtype AIV. And obviously the 8 genes of Ck/HB/03 did not show any close relationship with the isolated H9N2 subtype AIV from Korea, Iran, Pakistan, etc..From the above results, the pathologic agent of sick ducks from poultry raising farms in region of Yuyao was diagnosed as Duck parvovirus.And the research results of H9N2 subtype AIV,Ck/HB/03, indicated that the NA gene of Ck/HB/03 may take place the natural reassortment with Ck/HK/99 but the other 7 genes inclined to be more stable. Though the natural reassortment of the NA gene may exsist, Ck/HB/03 and other Chinese isolated viruses constructed a sublineage.