Isolation, Comparative Virulence And Mutation Trend Analysis Of Hemagglutinin Genes And Neuraminidase Genes Of16Avain Influenza A Viruses Subtype H9N2in Shandong Province

It has been30years since the first detection of H9N2subtype of avian influenza virusin China. Although H9N2subtype is a low pathogenic avian influenza virus, it has causedhuge economic losses due to the dysfunction of immune system and decrease eggproduction. It was well documented that H9N2subtype of avian influenza virus is the mostprevalent in chicken population in China Due to the role of the HA and NA genes in viralpathogenicity and host specificity of the virus, it is of great importance to carry out geneticanalyses of the HA and NA genes mutation of H9N2subtype avian influenza virus.1. Isolation, identification and analysis of HA and NA genes of H9N2subtype avianinfluenza virus.In the present study,16strains of H9N2subtype influenza virus was isolated andidentified from samples collected from different commercial broilers farms in Weifang,Yantai, Taiant Dongying and other regions in Shandong province during2010to2012.Phylogeneitic analysis was carried out after assembly of sequences HA and NA genes,translation of nucleotide sequence into protein sequence and initial multiple sequencealignments. The results revealed that HA genes of the16isolates has a96.3％～99.9％homology and the HA amino acid sequence of the isolates showed97.1％～99.6％identity.NA genes of the16isolates has a97.1％～99.6％homology and the NA amino acidsequence of the isolates showed95.1％～99.8％identity. In addition, phylogeneitic analysisof HA genes and NA genes revealed that all the16isolates belong to the same lineage,namely, Y280-like lineage. The amino acid motif of cleavage sites for all the sixteen virusesin the HA gene were RSSR↓GLF, which was consistent with the characterization of theLPAIV. Among these isolates, there exist7～9potential glycosylation sites in the HA aminoacid sequence. All the16isolates had a new glycosylation site NCS at position313. A newpotential glycosylation site, NNS and NNT respectively, was present in two of these viruses.Glycosylation site was eliminated at the position141and218in the other two viruses. HAreceptor-binding pocket analysis shows that the receptor binding sites were relativelyconservative except the198site and all the viruses at the position226have Leucine (L)amino acid which preferred human receptor2,6-linked sialic acid. Analysis of NA geneshowed stalk neuraminidase region of the16strains had a deletion of3amino acid residues(T, E, I) at positions63～65which corresponds to the molecular feature of H9N2circulatingin mainland China. In the present study, amino acid residues of hemadsorbing (HB) sites at the position366～373,368and402showed variation. However, amino acid residues at theposition431～433have not been changed. There were also4isolates with the samehemadsorbing (HB) sites as human-origin H9N2. The amino acid residues at the position69,86,146,200,234were highly conserved and showed no mutations. Glycosylation site waseliminated at the position264,331,402in some isolates, and10/16isolates was absent ofglycosylation site at the position402.Addtionally, a new glycosylation site at the position264was found in some isolates which corresponds to mutations in the NA gene of Shandongisolates. Amino acid residues of antigenic determinants showed active mutations at theposition328,368,402. The amino acid residues in the enzyme active sites of the isolateswere highly conserved and showed no mutations associated with resistance to the influenzaneuraminidase inhibitors such as oseltamivir and zanamivir.2. Comparative virulence of the16isolates of H9N2subtypeIn the second experiment, the virulence of16isolates was analyzed and compared bythe determination of the50%egg infectious dose (EID50), mean death time (MDT) ofchicken embryos, intracerebral pathogenicity index (ICPI), intravenous pathogenicity index(IVPI). The results showed that the EID50was between10-6.25/0.2mL and10-7.69/0.2mL, andthe MDT between91.6hours and105.9hours. The ICPI and the IVPI were zero whichcorresponds to the criteria set by WHO. The results of all the tests indicated that the16isolates are lowly pathogenic avian influenza viruses (LPAIV).