| Avian influenza is a respiratory disease in poultry caused by influenza A virus. Since first reportedin1878, the viruses have been pandemic all around the world. Both the highly pathogenic avianinfluenza virus and low pathogenic avian influenza virus remain a threat to veterinary and human publichealth.We isolated seven H5N1subtype avian influenza virus (AIV) from98wild duck specimens in Jilin,Shandong and Fujian province of China in2011. Phylogenetic analysis of the HA gene revealed thatfive viruses located in three provinces belong to the clade2.3.2.1, while two viruses collected fromShandong province belong to the clade2.3.4and7.2, respectively. The different branches of AIV showcommon pandemic in local areas of China. Sequencing analysis results show that the nucleotidehomology of HA genes of the seven viruses were88.2%-99.6%and nucleotide homology were above95%between the viruses of different clades and their representative strains. The viruses show a highnucleotide homology with the currently prevalent isolates in China. The sources of the internal genes ofA/wild duck/Shandong/1/2011, A/wild duck/Shandong/628/2011and A/wild duck/Jilin/ZF/2011arecomplicated which belong to recombinant viruses. Experimental infection of SPF chickens and ducksshow that three isolates have virulence to the SPF chickens and ducks, while the virus of the clade2.3.2.1has higher replication capacity in SPF chickens than that of the clade2.3.4and7.2.In order to explore the genetic mutations of the H9N2subtype AIV isolated in China, thesequences of the complete HA segments of the ten H9N2subtype AIVs were analyzed on homology andheredity evolution. The results show that the amino acid motif of cleavage sites for the viruses in the HAgene were PS/ARSSR↓GLF, which was consistent with the characterization of the low pathogenic avianinfluenza virus.7to9potential glycosylation sites were found in the HA gene and new potentialglycosylation sites were found at site127,148and295. The receptor binding sites were relativelyconservative except that of site190. The Leucine at the amino acid position226in the HA genes of sixisolates indicate the potential of binding with SAα-2,6receptor of mammals. The results indicate thatthe HA genes of the viruses displayed nucleotide homologies ranging from88.7%to99.8%. Theybelong to a branch of the A/Duck/Hong Kong/Y280/97in the phylogenetic tree. Based on the principleof5%homology diffence, we divided those viruses into four different subgroups: Y280-like-1,Y280-like-2, Y280-like-3and Y280-like-4. However, the viruses of Y280-like-1subgroup were thedominant strains in different areas of in China. Animal infection experiments show that the virusA/chicken/Beijing/15/2011belonging to Y280-like-1subgroup and A/chicken/Nanjing/17/2011belonging to Y280-like-4subgroup have higher virulence than that of Y280-like-2and Y280-like-3subgroups.To construct high-yield H9N2subtype AIV candidate vaccine strain propagating in MDCK cells bythe reverse genetics, A/chicken/Shanghai/11/2011(SH11) was chosen from ten isolates. The steady HItiter of1:1024of the rescued rSH11was achieved after5passages in MDCK cells. The virus rSH11andthe wild type virus shared similar biological properties such as50%embryo infective dose(EID50),50% tissue culture infective dose(TCID50) and genetic stability. Furthermore, the HI antibodies were detectedfrom SPF chickens immunized with oil-emulsified inactivated vaccine at one week postvaccination, andmore than1﹕700were achieved at three week postvaccination. The rSH11vaccine can dramaticallysuppress SPF chickens from shedding challenged virus. To construct high-yield AIV propagating inMDCK cells by the rSH11reverse genetics platform, the reassortment viruses replaced HA and NA ofrSH11by HA and NA of other viruses were rescued.In short, the study would provide basic data and information for H5N1and H9N2subtype AIV inChina. Meanwhile, the successful rescue of rSH11settles the foundation for the production of cellgrown influenza vaccine. |
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