禽流感病毒H5N1基因突变对神经氨酸酶抑制剂敏感性和神经氨酸酶活性的影响

目的 研究感染人的禽流感病毒H5N1神经氨酸酶突变I117V,I314V,I117V+I314V对神经氨酸酶抑制剂(NAIs)敏感性和神经氨酸酶(NA)活性的影响.方法 定点突变技术将变异位点引入禽流感病毒株A/Vietnam/1203/04(H5N1)(VN1203)的NA基因中,以A/WSN/33(H1N1)为背景,通过反向遗传学方法构建重组流感病毒,鸡胚传代增殖病毒,荧光定量评估病毒对NAIs的敏感性和NA活性(IC50值和Km,Ki值).结果 与野生株VN1203相比,I117V使NA对奥司他韦敏感性降低(IC50值升高了17倍,Ki值升高了20倍),对扎那米韦敏感性无明显影响(IC50值升高了2倍,Ki值升高了3倍),NA活性降低(Km值升高了23倍).I314V对NAIs敏感性和NA活性均无明显影响.结论 初步结果认为NA突变I117V和I314V对感染人的禽流感病毒H5N1并不产生NAIs耐药,临床上仍然可以用奥司他韦或扎那米韦抗病毒治疗.

Impact of avian influenza virus H5N1 neuraminidase mutations on the activity of neuraminidase and the sensibility to neuraminidase inhibitors

Objective To study the impact of avian influenza virus H5N1 neuraminidase mutations I117V,I314V and I117V+I314V on the sensibility of neuraminidase inhibitors(NAIs) and the activity of neuraminidase (NA).Methods The mutations were intreduced into NA genes of virus strain A/Vietnam/1203/04 (H5N1)by site-directed mutagenesis.With the A/WSN/33 (H1N1)background,recombinant influenza viruses containing NA mutations were rescued by reverse genetics.After viral propagation in chicken embryos,fluorimetric assays were conducted to assess the sensibility to NAIs and NA activity(IC50,Km&Ki).Results Compared to the wild-type viruS VNl203.the mutation I117V decreased the susceptibility to osehamivir(17-fold increment of IC50 value,20-fold increment of Ki value)and the NA activity(23-fold increment of Km value)while there Was little impact on zanamivir sensitivity(2-fold increment of IC50 value,3-fold increment of Ki value).The mutation 1314V had no marked influence on either the NA activity or the NAIs susceptibility.Conclusion It aDpears that the NA mutations of I117V and I314V Can not cause NAIs resistanee.Oseltamivir or zanamivir may still be prescribed for anti-viral treatment.