| Since first reported in China form 1994, H9N2 avian influenza had spread to most regions of China. In recent years the separation reports on H9N2 subtype AIV showed a rising trend and resulted in huge economic losses in poultry industry. Furthermore H9N2 avian influenza virus also donated its internal gene to the newly emerging H7N9 and H10N8 avian influenza virus which can infect human, potential threats to human health and safety. Thus study on the genetic variation and antigenic evolution of H9N2 avian influenza virus is of important economic and public health significance.To clarify the epidemic variation of H9N2 subtype AIV in China, forty-two H9N2 subtype AIV strains were isolated from vaccinated chicken farms in China from 2012 to 2015, performed whole genome sequencing of H9N2 AIV strains and constructed phylogenetic trees. Cross HI test is token by the single factor serum of eighteen representative H9N2 strains to evaluat the antigenic evolution among H9N2 epidemic strains. The protective effect of Ck/GX/SIC6/13 on the epidemic strains was evaluated by immune protection test.Phylogenetic anylasis shows: 42 H9N2 AIV strains were belong to Ck/Bei-like lineage, 97.6%(41/42) of the isolates were classified to h9.4.2.5 clade, and 2.4%(1/42) of the isolates were classified to h9.4.2.6 clade. Their HA genes had nucleotide sequence homology from 86.7% to 99.7%, while similarity to the classic vaccine strain was 88.6%~92.6%. The isolates were divided into four different genotypes(S, U, V and W). Genotype S was the major one, accounted for 92.8%(39/42). Genotypes V and W were new reassortment genotypes, with genotype W recombined with the PB2 gene originated from the new wild waterfowl-like lineage. Our result shows the gene recombination of H9N2 virus became more complicated.Specific amino acids site analysis indicated that, 97.6%(41/42) of the isolates possessed PSRSSR?GLF motif at the cleavage site of the HA protein, except one strain possessed PARSSR?GLF motif, are the characteristics of low pathogenic avian influenza viruses; 92.8%(39/42) HA genes showed human-receptor-binding-associated mutation Q226?L. A total of 95.2%(40/ 42) of the viruses had three amino-acid deletions at 63-65 in NA stem, associated with enhanced virulence; 97.6%(41/42) M2 proteins had the S31?N mutation associated with adamantane resistance. In our study only 2.4%(1/42) of the strains appeared E627?K mutations in PB2 proteins, and no D701?N mutations were detected in PB2 proteins.Cross HI test data and R values shows: Single factor antisera of the 18 new isolates had 0~9 log2 HI titer difference against homologous and heterologous viral antigens, antigenic relevance(R values) of the 18 new isolates is 0.41~1, even if H9N2 viruses origin form the same lineage, its antigenicity is also gradually differentiation. Based on phylogenetic anylasis and cross HI test, the new isolates could be divided into three antigenic groups(A, B and C), analysis of isolation times, places and evolutionary relationships showed that appearance of the three antigenic groups roughly followed the order of A?B?C.Immune protection test shows that the inactivated vaccine prepared with early strain of H9N2 viruses can not provide effective protection, protection rate to the viruses of A group is only 30% ~ 50%; and the inactivated vaccine prepared with Ck/GX/SIC6/13 can only protect the challenge of homologous virus, but unsuitable for heterologous viruses. Suggested that the antigenic variation of H9N2 epidemic isolates in recent year is more frequently, the antigenicity and cross-protection among the H9N2 epidemic isolates is more complexity and weakly. |
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