| Avian influenza(AI) is a kind of infectious diseases caused by AIVs, which can be classified into three pathotypes based on pathogenicity, named highly pathogenic AI, low pathogenic AI and non-pathogenic AI. H9N2 subtype AI is the main subtype of low pathogenic AI. In China, the unique geographical environment and breeding patterns provide favorable condition for the occurrence and transmission of this disease, with the addition of the continued immune pressure of vaccine and trade in live poultry provide a suitable condition for recombination and variation of AIV.In order to monitor the antigen variation of H9 subtype AIV in poultry, cloacal swabs collected from live bird markets (LBMs) and specimens collected from diseased poultry during 2013-2015 in Eastern China were used to isolate AFVs.The whole genome of H9N2 isolates were sequenced and phylogenetic analysis were performed.We not only evaluated antigenic differentence of the dominant strains and these epidemic strains but also the currently widely used F98 strain in East China. In addition, Monoclonal antibodies (McAbs) against H9N2 subtype AIV strain from WXQ3 (G1-Like) was also developed. The aim of this study is to understand the genetic variation and antigenic differences, which to provide experimental basis for prevention and control of avian influenza.1. Epidemiological investigation and phylogenetic analysis of H9N2 subtype AIV in Eastern China from 2013 to 20154500 cloacal swabs from two LBMs and 305 specimens from diseased poultry were collected from Jiangsu province and surrounding areas during 2013-2015.35 strains of H9 subtype AIV were isolated by inoculating embryonated SPF eggs and identified by HA and HI test combined with RT-PCR. The isolation rate of H9 AIV from cloacal swabs was 0.75%, and that from specimens was 0.33%. The isolation rate of AIVs from chickens was higher than other animal.Phylogenetic analysis for nucleotide sequences of HA genes in the 35 H9N2 AIVs indicated that these AIVs were all classified in Y280-Like (Clade h9.4.2), which became the predominant strains in Eastern China(consistent with previous research data in our this lab). But there were mutations among these viruses, in which the nucleotide homology of HA genes was 89.8%-91.8%. The residues 226 and 228 in HA protein of all H9N2 AIVs were Q and G, respectively, indicating a preference to bind to avian cell receptor.NA genes of all H9N2 AIVs in this study belonged to Y280-Like, in the position 63-65 of the NA stalk three amino acid were deleted. Genotype analysis insisted that these genotypes of H9N2 isolates belonged to a kind of V genes, HA and NA genes from Y280, PB2, and M genes from G1, PB1, PA, NP, and NS genes from F98.2. Different antigen analysis of predominant strains H9N2 subtypes AIVsTwo H9N2 influenza viruses were selected from local epidemic strains, named JSWX and JSYZ110, which belonged to different subtree by using antigenic analysis and cross hemagglutination inhibition test based on phylogenetic analysis. Then, SPF chickens were immunized with inactivated JSWX, JSYZ110, and F98. On 21 days after immunization, the chickens were inoculated intranasally with 106 EID50 doses of homologous and heterogonous avian influenza H9N2 virus. The results showed that the rates of viral shedding of the JSYZ110 group and JSWX group both were 3/10, but that of the F98 group was 4/10 not only against the homologous H9N2 AIV challenge, but also against heterologous H9N2 AIV challenge, proving that there were no antigen differences between the epidemic strains, while there were antigen differences between the epidemic strains and the F98 strain.3. Development of monoclonal antibodies against H9N2 subtype AIVs of G1-LikeOne representative AIV strains WXQ3 belongs to G1-like was chosen as antigen to prepare the McAbs. Viruses purified by differential centrifugation were used to immunize 6-8 weeks old Balb/c mice. Positive clones were screened by haemagglutination inhibition (HI), and five monoclonal antibody hybridoma cell lines were obtained, named as 2E4,4D10,8B2,3F6, and 10G8, respectively, which titer were 25 27 23 24, and 24. Specificity results showed that all five McAbs had no reaction with H5 AIV, H7 AIV, NDV, and IBV. The results of HI showed that the McAb 2E4 had HI titer against H9N2 AIV strain WXQ3, HN and JSYZ110, these McAbs 4D10, 3F6, and 10G8 had HI titer against H9N2 AIV strain WXQ3, HN, F98, JSYZ110, and AHFY040, the McAb 8B2 had HI titer against H9N2 AIV strain WXQ3,HN, and AHFY040.The results of Western-blot showed that three McAbs named 4D10,8B2, and 3F6 from WXQ3 reacted with AIV stain WXQ3, all with a 50-kDa specific band. The results of indirect immunofluorescent assay revealed that all the three McAbs named 4D10,8B2, and 3F6 had specific fluorescent reaction to AIV strain WXQ3, the McAb named 2E4 had specific fluorescent reaction to AIV strain HN, all the five McAbs had specific fluorescent reaction to ATV strain F98.The result of cross-reactivity HI showed that McAbs named 10G8,3F6, and 4D10, had a broad spectrum of haemagglutination inhibition against H9 AIV. |
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