| H9N2 subtype avian influenza virus (AIV) belongs to low pathogenic avian influenza virus, but it can cause severe disease, especially high mortality in mixed infection. The virus can also be a donor for other subtype influenza virus to produce new recombinant virus. Currently, vaccine immunization is the major method for preventing and controlling of H9N2 AIV. However, the speed for vaccine development can’t match up with the rapid speed of antigen variation of AIV in the process of prevail and evolution. Therefore, it is necessary to develop more sustainable and effective vaccine. Cold-adapting is a quite secure way to attenuate virus, which not only preserves antigenicity, but also induces high-level immune response. In this study, we cultivated a cold-adapted attenuated vaccine using a H9N2 subtype avian influenza virus, constructed the reverse genetic system of the cold-adapted (Ca) vaccine to identify the genes for controlling of Ca phenotype and evaluated its immune efficacy. This study laid a foundation for the development of new avian influenza vaccines.1 Development and biological characteristics of cold-adapted live attenuated strain of H9N2 AIVA strain of cold-adapted live attenuated H9N2 influenza viruses was obtained by adaptation of the virus A/chicken/TaiXing/1/2008(TX) in chicken embryonated eggs through passaging with progressive decreasing temperature. The virus was passaged for 8-10 times at each temperature. Finally, the virus could replicate stably at 25℃. The cold-adapted virus was proved to have Ca phenotypes and to replicate well at 33 ℃ in experiments. In order to analysis amino acid changes in cold-adapted progress,8 genes of cold-adapted strain were amplified by PCR and sequenced. The result showed that each gene had varied degrees of mutants and mutants mainly occurred in HA, PB2, PB1, PA, NP, compared with parent strain. In vitro proliferation assay showed that cold-adapted strain replicated well in CEF, but couldn’t replicate in MDCK. At same time, it wasn’t capable of replicating in mice lungs and resulted in body weight loss of mice. The data indicated that the virulence of cold-adapted virus was obviously reduced through the adaptation.2 Immune efficacy of cold-adapted live attenuated strain of H9N2 AIVTo evaluate the potential of cold-adapted strain of H9N2 AIV as vaccine candidate, chickens were inoculated with 106 EID50 of virus by intranasal and intraoral routine. The result showed that the cold-adapted virus lost contact-transmissibility within chickens and TX-25-CE30 was only able to replicate in trachea tissues at low level. When 4-week-old SPF chickens were intranasally inoculated with 106 EID50 of viruses or injected intramuscularly with inactivated vaccine of same dosage as control group, only rTX-NS 1-128 induced high-level HI antibody (8.45 log2) and provided 80% protection rate after one-dose vaccination against homologous (TX) and heterologous (F98) H9N2 viruses challenge. On the other hand, compared with immune efficacy of rTX-NS 1-128, TX-25-CE30 and inactivated vaccine provided 60% protection rate and induced HI antibody (7.0 log2) in chickens with one-dose vaccination. It had better efficacy in chickens with a second vaccination.3 Determination of genes for controlling the cold-adapted phenotype of the cold-adapted H9N2 AIVTo identify relevant critical cold-adapt genes of cold-adapted strain of H9N2 AIV, eight genes of the strain were amplified, and cloned into pHW2000 to construct eight plasmid systems. The eight plasmid systems of PR8 combined with substitution of single gene fragment or two gene fragments of cold-adapted vaccine were cotransfected into MDCK and 293T cells to obtain 12 recombined viruses. The result of Ca phenotype assay showed that the NP, PB1 and PB2 fragments were responsible for the Ca phenotype, and NP gene played a critical role for the Ca phenotype. For further determination of the amino acids of NP segment, the result showed that G18E in NP gene was sufficient to allow virus to grow at 25℃. |
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