Research Center in Infectious Diseases of the CHUQ-CHUL and Laval University, Québec City, Québec, Canada.
Antimicrob Agents Chemother. 2012 Mar;56(3):1208-14. doi: 10.1128/AAC.05994-11. Epub 2011 Dec 27.
Amino acid substitutions at residue I223 of the neuraminidase (NA) protein have been identified in 2009 pandemic influenza (pH1N1) variants with altered susceptibilities to NA inhibitors (NAIs). We used reverse genetics and site-directed mutagenesis to generate the recombinant A/Québec/144147/09 pH1N1 wild-type virus (WT) and five (I223R, I223V, H275Y, I223V-H275Y, and I223R-H275Y) NA mutants. A fluorimetry-based assay was used to determine 50% inhibitory concentrations (IC(50)s) of oseltamivir, zanamivir, and peramivir. Replicative capacity was analyzed by viral yield assays in ST6GalI-MDCK cells. Infectivity and transmission of the WT, H275Y, and I223V-H275Y recombinant viruses were evaluated in ferrets. As expected, the H275Y mutation conferred resistance to oseltamivir (982-fold) and peramivir (661-fold) compared to the drug-susceptible recombinant WT. The single I223R mutant was associated with reduced susceptibility to oseltamivir (53-fold), zanamivir (7-fold) and peramivir (10-fold), whereas the I223V virus had reduced susceptibility to oseltamivir (6-fold) only. Interestingly, enhanced levels of resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir (1,647-, 17,347-, and 16-fold increases in IC(50)s, respectively) were observed for the I223R-H275Y recombinant, while the I223V-H275Y mutant exhibited 1,733-, 2,707-, and 2-fold increases in respective IC(50)s. The I223R and I223V changes were associated with equivalent or higher viral titers in vitro compared to the recombinant WT. Infectivity and transmissibility in ferrets were comparable between the recombinant WT and the H275Y or I223V-H275Y recombinants. In conclusion, amino acid changes at residue I223 may alter the NAI susceptibilities of pH1N1 variants without compromising fitness. Consequently, I223R and I223V mutations, alone or with H275Y, need to be thoroughly monitored.
神经氨酸酶（NA）蛋白 223 位的氨基酸取代已在对神经氨酸酶抑制剂（NAI）敏感性改变的 2009 年大流行性流感（pH1N1）变异株中被鉴定。我们使用反向遗传学和定点诱变生成重组 A/魁北克/144147/09 pH1N1 野生型病毒（WT）和 5 个（I223R、I223V、H275Y、I223V-H275Y 和 I223R-H275Y）NA 突变体。使用荧光测定法测定奥司他韦、扎那米韦和帕拉米韦的 50%抑制浓度（IC（50））。通过 ST6GalI-MDCK 细胞中的病毒产量测定分析复制能力。在雪貂中评估 WT、H275Y 和 I223V-H275Y 重组病毒的感染性和传播性。如预期的那样，与药物敏感的重组 WT 相比，H275Y 突变赋予了奥司他韦（982 倍）和帕拉米韦（661 倍）的耐药性。单一的 I223R 突变与奥司他韦（53 倍）、扎那米韦（7 倍）和帕拉米韦（10 倍）的敏感性降低有关，而 I223V 病毒仅对奥司他韦（6 倍）的敏感性降低。有趣的是，对于 I223R-H275Y 重组体，观察到奥司他韦和帕拉米韦的耐药性增强以及扎那米韦的敏感性降低（IC（50）分别增加 1734 倍、17347 倍和 16 倍），而 I223V-H275Y 突变体则表现出 1733 倍、2707 倍和 2 倍的相应 IC（50）增加。与重组 WT 相比，I223R 和 I223V 变化在体外与更高的病毒滴度相关。在雪貂中，重组 WT 与 H275Y 或 I223V-H275Y 重组体之间的感染性和传播性相当。总之，氨基酸 223 位的改变可能会改变 pH1N1 变异株对 NAI 的敏感性，而不会影响适应性。因此，需要彻底监测 I223R 和 I223V 突变，以及单独或与 H275Y 一起。
