Escuret Vanessa, Collins Patrick J, Casalegno Jean-Sébastien, Vachieri Sebastien G, Cattle Nicholas, Ferraris Olivier, Sabatier Murielle, Frobert Emilie, Caro Valérie, Skehel John J, Gamblin Steve, Valla Frédéric, Valette Martine, Ottmann Michèle, McCauley John W, Daniels Rodney S, Lina Bruno
Laboratoire de Virologie et Centre National de Référence virus influenzae Laboratoire Virpath EA4610, Faculté de Médecine Lyon Est, Université Claude Bernard Lyon 1, Université de Lyon, and.
Division of Virology.
J Infect Dis. 2014 Oct 15;210(8):1260-9. doi: 10.1093/infdis/jiu244. Epub 2014 May 3.
Influenza B viruses with a novel I221L substitution in neuraminidase (NA) conferring high-level resistance to oseltamivir were isolated from an immunocompromised patient after prolonged oseltamivir treatment.
Enzymatic characterization of the NAs (Km, Ki) and the in vitro fitness of viruses carrying wild-type or mutated (I221L) NA genes were evaluated. Proportions of wild-type and mutated NA genes were directly quantified in the patient samples. Structural characterizations by X-ray crystallography of a wild-type and I221L variant NA were performed.
The Km and Ki revealed that the I221L variant NA had approximately 84 and 51 times lower affinity for oseltamivir carboxylate and zanamivir, respectively, compared with wild-type NA. Viruses with a wild-type or I221L variant NA had similar growth kinetics in Madin-Darby canine kidney (MDCK) cells, and 5 passages in MDCK cells revealed no reversion of the I221L substitution. The crystal structure of the I221L NA and oseltamivir complex showed that the leucine side chain protrudes into the hydrophobic pocket of the active site that accommodates the pentyloxy substituent of oseltamivir.
Enzyme kinetic and NA structural analyses provide an explanation for the high level of resistance to oseltamivir while retaining good fitness of viruses carrying I221L variant NA.
从一名免疫功能低下患者在接受长时间奥司他韦治疗后分离出了在神经氨酸酶（NA）中具有新型I221L替换且对奥司他韦具有高水平耐药性的乙型流感病毒。
评估了NA的酶学特性（Km、Ki）以及携带野生型或突变型（I221L）NA基因的病毒的体外适应性。直接对患者样本中的野生型和突变型NA基因比例进行了定量。对野生型和I221L变异型NA进行了X射线晶体学结构表征。
Km和Ki显示，与野生型NA相比，I221L变异型NA对奥司他韦羧酸盐和扎那米韦的亲和力分别低约84倍和51倍。携带野生型或I221L变异型NA的病毒在Madin-Darby犬肾（MDCK）细胞中具有相似的生长动力学，并且在MDCK细胞中传代5次后I221L替换未发生回复突变。I221L NA与奥司他韦复合物的晶体结构表明，亮氨酸侧链伸入容纳奥司他韦戊氧基取代基的活性位点的疏水口袋中。
酶动力学和NA结构分析为携带I221L变异型NA的病毒对奥司他韦具有高水平耐药性同时保持良好适应性提供了解释。
