流感病毒H3N2神经氨酸酶双点突变对病毒抗药性影响的研究

摘要

To determine the key sites responsible for drug resistance in H3N2 neuraminidase gene, H3N2 wild type and three mutation type (E119D+I222L,E119D and I222L) viruses were generated by reverse genetics; The reverse genetics viruses were stable after five passages in MDCK cells; Oseltamivir resistance was detected by analyzing the kinetics of the two types of viruses in MDCK cells in the absence or in the presence of oseltamivir at the indicated times. We successfully generated H3N2 wild type and mutation type viruses; The wild type had a similar virus titer with the mutation type E119D; The virus titer of E119D+I222L and I222L type were lower than the wild type; The mutation type E119D+I222L had a high oseltamivir resistance while the wild type, E119D type and I222L type were greatly inhibited by oseltamivir. These results indicated that double mutation (E119D+I222L) type H3N2 could induce a high level of oseltamivir resistance. Thus, two amino acids in H3N2 neuraminidase gene (119, 202) are the two key sites responsible for oseltamivir resistance.%为验证流感病毒H3N2神经氨酸酶上2个抗药性的关键位点,利用反向遗传学手段,8个质粒共同转染293T细胞,包装带有双点突变(第119位氨基酸由E突变为V,第222为氨基酸由I突变成L)、单点突变和野生型的流感病毒H3N2;在MDCK细胞中传代包装成的H3N2病毒,检测不同感染时间和有无抗流感病毒药物(奥司他韦)存在下病毒的滴度.结果表明,野生流感病毒H3N2和其突变株包装成功,第119位氨基酸单点突变型滴度与野生型的相近,而第222位氨基酸单点突变和双突变型滴度要比野生型的低；双点突变株相比野生株具有很高的抗奥司他韦的生物活性,而单点突变型不具有抗药活性.本试验成功证明了H3N2神经氨酸酶上2个位点(第119和222位氨基酸)对病毒抗药性起到关键的作用,且只有双点同时突变的条件下,病毒才会有抗奥司他韦的生物活性.