高增殖猪流感重组病毒GX1659/PR8的制备与鉴定

Development and identification of high-replicated Swine influenza virus GX1659/PR8 reassortant virus

为构建一株具有高复制能力的流感病毒疫苗株,本研究利用反向遗传技术以PR8的6个内部基因为骨架,以一株H3N2亚型低致病力猪流感病毒(SIV)GX1659株的HA和NA表面基因为供体,构建了pBD-GX1659-HA和pBD-GX1659-NA重组质粒,转染293T细胞并接种SPF胚,经血凝试验、测序筛选获得了一株重组病毒GX1659/PR8。该重组病毒在鸡胚传20代,血凝价均能达到8 log2以上。每5代病毒的全基因组经PCR和测序鉴定,结果显示,PCR产物均无任何缺失或突变,表明GX1659/PR8具有遗传稳定性。分别利用100 EID_(50)剂量的GX1659和GX1659/PR8感染鸡胚,在不同时间测定鸡胚尿囊液的血凝效价和EID_(50),绘制其生长曲线。结果显示,GX1659和GX1659/PR8分别在接种后60 h和48 h时血凝效价达到峰值,GX1659/PR8效价滴度明显高于亲本病毒GX1659。利用亲本病毒GX1659与重组病毒GX1659/PR8的抗原和阳性血清进行交叉HI试验,测定HI效价及交叉HI效价并对抗原性进行分析。结果显示,亲本病毒GX1659与重组病毒GX1659/PR8抗原性无明显差异。本研究拯救出一株具有稳定遗传性和高增殖性能的H3亚型重组病毒GX1659/PR8,为H3N2亚型流感病毒疫苗株的构建奠定了重要基础。

In order to develop a vaccine candidate strain of Influenza virus with good proliferation ability, the recombinant plasmids of pBD-GX1659-HA and pBD-GX1659-NA was constructed expressing hemagglutinin(HA) and neuraminidase(NA) of low virulence swine influenza virus GX1659, respectively generated by using reverse genetics system based on six internal genes of PR8 backbone. They were used to transfected 293 T cells, and the obtaining recombinant virus GX1659/PR8 was identified by hemagglutination(HI) test and sequencing. The hemagglutination titer of the recombinant virus was more than 8 log2 in 20 generations of chicken embryos. The PCR amplification and sequencing of the whole genome were performed for every 5 generations of the virus. The results showed that there were no deletions or mutations in PCR amplicons, indicating that GX1659/PR8 had inheritable stability. SPF embryos were infected with GX1659 and GX1659/PR8 containing 100 EID_(50), and virus samples were taken at different periods to determine the HA titer and EID_(50)value and the viral growth curves were drawn. The results showed that the HA titers of GX1659 and GX1659/PR8 reached their peaks at 60 hours and 48 hours after inoculation, and the HA titer of GX1659/PR8 was significantly higher than that of the wild-type virus GX1659. The antigens and anti-sera of parental virus GX1659 and recombinant virus GX1659/PR8 were used for cross-HI tests. The titers of HI and cross-HI were determined and the antigenicities of both viruses were compared. The results showed that there was no significant difference in antigenicity between parent virus GX1659 and recombinant virus GX1659/PR8. In this study, a H3 subtype recombinant virus GX1659/PR8 was rescued, with stable genetic characteristics and high proliferation performance, which provided an important foundation for the development of H3 N2 subtype swine influenza virus vaccine.