H1N1亚型猪流感病毒反向遗传操作系统的建立

 【目的】建立H1N1亚型猪流感病毒反向遗传学操作系统及拯救出能够在动物传代细胞中高水平复制的H1N1亚型猪流感疫苗株。【方法】利用反向遗传操作技术，对猪流感病毒广东分离株进行拯救。【结果】首次成功拯救出全部片段均来自于亲本株的猪流感病毒rH1N1，并且成功拯救出了具有高度细胞适应性毒株re-LM株，研究结果表明二者均具有良好的遗传稳定性。rH1N1经MDCK细胞连续传代培养后，血凝价最高仅为1﹕64;而re-LM的血凝价最高可以稳定在1﹕1 024，表明该毒株具有细胞繁殖高产的特性。用该重组病毒制备油乳剂灭活苗免疫2月龄仔猪，首免2周即可检测出HI抗体，平均效价在1﹕32以上，三免后2周HI抗体平均效价达到1﹕512，说明其有良好的免疫原性。【结论】H1N1亚型猪流感病毒反向遗传操作系统的成功建立为猪流感病毒致病机理、传播机制及病毒基因功能的研究奠定了基础;重组细胞高产型猪流感病毒株的拯救为H1N1亚型猪流感疫苗的研制开辟了新的途径。

Establishment of Reverse Genetics of H1N1 Subtype Swine Influenza Virus and Generation of Cell Culture High-Yield Recombinant Vaccine Candidate

【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.