关键词: 猪流感, H1N1亚型, 反向遗传操作技术, 细胞培养型流感疫苗

Abstract:

【Objective】 To establish a reverse genetics system of H1N1subtype swine influenza virus and to construct high-yield H1N1 subtype swine influenza virus candidate vaccine strain propagating in MDCK cells. 【Method】 A/Swine/ Guangdong/LM/2004 (H1N1) was chosen to generate new recombinant virus by reverse genetics. 【Result】 rH1N1 was rescued, and all the genes of which derived from the parental strain. After several rounds passages of the rH1N1 in MDCK cells, maximum HA titer of 1﹕64 was achieved. Meanwhile, re-LM was obtained which contained hemagglutinin (HA) and neuraminidase (NA) genes derived from LM, and the other six internal genes from A/Goose/Dalian/3/01(H9N2). Both rH1N1 and re-LM showed a perfect stability. The maximum HA titer of re-LM was 1﹕1 024 was observed, after consecutive passages in MDCK cells, moreover, the HA titer was steady, which showed that re-LM had the characteristics of high-yield. A formalin- inactivated oil-emulsion vaccine was prepared with re-LM. Inoculation of piglets with the inactivated vaccine, the HI antibodied were detectable at two weeks post-vaccination. The average HI titers were above 1﹕32 after two weeks of the first vaccination, and which could reach 1﹕512 after the third times vaccination, demonstrating it′s optimal immunogenicity. 【Conclusion】 This is the first report of successful in establishment of the reverse genetics system of H1N1 subtype swine influenza virus in the world. It settles the foundation for further research on pathogenicity mechanism and the production of cell grown influenza vaccine of H1N1 swine influenza in the future.

Key words: swine influenza, H1N1 subtype, reverse genetics, cell grown influenza vaccine