| H9N2low pathogenic avian influenza (LPAI) disease is one of the important epidemic diseases that can harm commercial broilers, laying hens and breeder chickens and one of the AIV subtypes which is currently the most widely popular and harmful to chicken flocks. Since this disease first occurred in our country in1992, it has become a common disease in chickens. This study evaluated the antigenic change of H9N2comprehensively by pathogenicity, hemagglutination inhibition test, chicken embryos neutralization experiment and vaccine protective efficacy trials. From the perspective of whole genome, the characteristics of H9N2gene rearrangement in recent years were analyzed, especially the genetic variation of hemagglutinin (HA) in the year1999to2013. Moreover, this research also studied the influence and correlation between the genetic variation and the virus antigenicity. The progress listed as follows:1. Isolation, identification and biological characteristics determination of H9N2Shandong strain35strains of H9N2excluding exogenous viral infections were isolated and identified in diseased flocks in Shandong province (19strains were isolated before2010and16strains in2010-2013). The final concentration dilution method was used to virus purification and biological virulence measurement. The EID50per0.1ml of strains which isolated after2010were between107.83to109.20, the IVPI result was0to0.17. Compared with the separation of the laboratory from1999to2010H9N2strain, the virulence has increased and should be alert to the enhanced virulence trend of H9N2.2. The analysis of viral whole genome of H9N2The purified viral HA genes were sequenced reference to the classical methods. Then the homologous analysis and molecular characterization comparison were also studied. The results showed that all35AIVs which were isolated and identified from1999to2013in Shandong belonged to the Eurasian lineage, including BJ94-like (3strains,1996-2005), Y280-like (4strains,1999±2009), S2-like (28strains,2010～2013), which indicated the major epidemic strains in Shandong province in recent years was S2-like subgroup of H9N2. The nucleotides (amino acid) homology of16strains found after2010was94.5%to100%(96.1%to100%), but the homology of the classical vaccine SD-1996’s has been reduced homology to90.0%-92.5%(91.8%to95.0%). The analyses of proteolytic cleavage site showed that, its proteolytic cleavage site motifs mostly was PARSSRJ,GL during the years1994-2010, while after2010, the majority were PSRSSR↓GL, which was still meeting the sequence features of low pathogenic avian influenza and consistent with the biology of virulence detection index.The whole genome sequencing of8epidemic strains chose from different isolated time showed that, all strains belong to the Eurasian lineage. Compared to the HA and NA genes, the NS gene was conservative and the genetic evolution of other6encoding proteins displayed diversity. Phylogenetic analysis showed that the PB2gene and M gene of CK/SD/DY/2009, CK/SD/SIX/2010, CK/SD/T07/2010and CK/SD/HKY1/2013were all derived from the G1-like, the PB2gene of CK/SD/BD/2008came from the F98-like and M gene was from the G1-like; and early isolates and reference strains were all derived from BJ94-like or F98-like. The genotypes of strains before2005were BJ94-like strains, whereas the PB1, PA, NP gene of strains after2008belong to F98-like, the NA, NS gene belongs to BJ94-like, and M gene belongs to G1-like, but there were changes appeared in gene PB2and HA which was F98-like, G1-like and BJ94-like and S2-like. This indicated that there existed varying degrees of rearrangements in the eight fragments of H9N2subtype avian influenza virus strains, which resulting in multiple genotypes of H9N2virus.3. The analysis of H9N2virus hemagglutinin antigenicityAccording to the result of the gene analysis of HA,8representative viruses from the different genetic evolution branches were chose to HI cross-inhibition experiment.The results showed that homologous virus titer was higher on its hemagglutination inhibition, while the crossing HI was relatively lower between the H9N2in different periods. This indicated that H9N2had a great degree of variation on the antigenicity compared to the classic vaccine strain in China. At the same time, further statistical analysis showed that there was a significant correlation of homology of H9N2subtype AIV virus HI related index and HA gene amino acid(p<0.05), Y=-3.53X+2.4487, R=0.6398.which indicated that genetic variants were mainly associated with the HA gene of H9N2subtype. Chicken embryo neutralization test also proved that.4. Vaccine protective efficacy trialsVaccine protective efficacy trials showed that, compared with the classic vaccines, the clinical symptoms after infection, virus isolation rate and detoxification of vaccine prepared with pandemic strain were significantly declined, the protective rates of SD/96vaccine for YZ/00and JN/05were80%, but for the protection of epidemic strains of LC02/13rate was only40%; while the protection rate of LC02/13inactivated vaccine for the4strains were100%, which demonstrating the vaccines prepared with the new pandemic strains has more effective protective effects.In summary, this paper had carried system analysis and research on the genetic evolution and the antigenicity of H9N2in Shandong and also evaluated the immune effects, the research results provided theoretical basis for the prevention and control of H9N2subtype avian influenza in Shandong province. |
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