The Isolation, Identification And Polymerase Activity Of Duck H5N5 Avian Influenza Virus

Avian Influenza(AI) is a viral infectious disease which caused by influenza virus A(AIVs). AIVs are divided into lots of several levels, 17 HA subtypes and 10 NA subtypes have been identified. Almost all AIVs subtypes can infect poultry, However, the H1 H3 H5 H6 H7 H9 subtypes can infect the human, and H5 subtype is the most common. Because of the limitation of the host range, AIVs type A rarely infects humans.However, in recent years, some diseases has been increased by the H5N1 subtype of AI. Although there are no reports about AIVs infection persistent in humans, the continuous antigenic drift may be caused by the environmental selection pressure. AIVs may crossed the species barrier to infect humans because of the adaptive mutation and the continuous antigenic drift of H5 subtype AIVs. Therefore, it is very important to strengthen the research on pathogenicity and transmission of H5 subtypes AIVs.In this study, we collected the case of the dead ducks, and then was inoculated into 10-day-old SPF chicken embryos after conventional treatment, allantoic fluid was collected of chicken embryo died within24-96 h. Hemagglutination assa(HA), hemagglutination inhibition(HI) test, the RT-PCR identification, and nucleotide sequence analysis of HA and HN were conducted. The results showed that the virus was identified as a H5N1 subtype avian influenza viruses and named A/Duck/Changchun/01/2010. Gene evolution analysis of HA and NA indicated that the strain belongs to new H5N1 influenza virus 2.3.2.1 strain, which prevalent in east Asia. The study results provided important information for the further study of molecular evolution on H5N1 subtype avian influenza virus from the ducks.In order to further study the evolution features of the isolated strains, specific primers of amplification nucleoprotein(NP) and polymerase protein(PB2 PB1 PA) gene were designed and synthesized, reverse transcription and PCR amplification were completed for the nucleoprotein(NP) and polymerase protein(PB2PB1 PA) gene, At the same time, gene sequences were analysed though bioinformatics software or on-line analysis software. The signal peptide transmembrane region and hydrophobic and predict potential glycosylation sites and phosphorylation site were analyzed. There was high homology of amino acid sequence between the strain and the major epidemic in South China waterfowl AIVs.The mutations at the 627, 701 AA in the PB2 protein of the AIVs play an important role in the host adaptability of influenza viruses, in order to study the polymerase activity of the duck H5N5 subtype AIVs and the changement after the amino acid mutations in the 627 th and 701 th amino acid. In this study, the amino acid mutations technology was concomitantly inserted in PB2. Restriction site was designed in view of polymerase gene sequence design, four plasmids by enzyme digestion were connected to expression vector,the point mutations were inserted in PB2 1 879, PB2 2 101, PB2 1 879 and 2 101 by the PCR method, Thepb2genesinthe627aminoacidsoftheaivswerechangedfromglutamicacid(glu)tolysine(lys),andthegenesinthe701aminoacidsfromasparticacid(asp)toaspartyl(asn).recombinantplasmidsweretransfectedinto293 tcellsbyaliposomemediatedtransfectionmethod,adual-luciferasereporterassaysystemwasusedtoinvestigatetheinfluenceofthesemutationsontherdrpactivity.thesefindingsindicatethatthresholdfluorescenceofpolymeraseinthepb2proteinoftheduckh5n5viruses(a/duck/changchun/01/2010)islower.thereislittleinfluenceonthepolymeraseactivitythroughthereplacementpb2proteincontainingmutationinthe2101 bases,thepolymeraseactivitywasincreasedthroughthereplacementpb2proteincontainingmutationinthe1879bases.however,thepolymeraseactivitywassignificantlyincreasedthroughthereplacementpb2proteincontainingmutationinthe1879and2101 bases.