Cell Culture-Selected Substitutions in Influenza A(H3N2) Neuraminidase Affect Drug Susceptibility Assessment
Cell Culture-Selected Substitutions in Influenza A(H3N2) Neuraminidase Affect Drug Susceptibility Assessment
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DOI：
10.1128/aac.01364-13
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发表时间：
2013-12-01
影响因子：
4.9
通讯作者：
Gubareva, Larisa V.
中科院分区：
文献类型：
--
作者：
Tamura, Daisuke;Nguyen, Ha T.;Gubareva, Larisa V.
药物敏感性评估已成为流感病毒监测的一个组成部分。在这项研究中，我们描述了A/密西西比/05/2011型流感病毒（H3 N2）的耐药性概况，该病毒从接受奥司他韦治疗的患者中收集，并通过监测检测。在神经氨酸酶（NA）抑制试验中，该病毒的MDCK细胞生长分离株显示出神经氨酸酶（NA）抑制剂（NAI）奥司他韦（8，005倍）、扎那米韦（813倍）、帕拉米韦（116倍）和拉尼米韦（257倍）的抑制作用高度降低。其NA基因的序列分析揭示了一个已知的奥司他韦抗性标记，在位置119处的谷氨酸到缬氨酸的取代（E119 V），和另外的改变，在位置148处的苏氨酸到异亮氨酸（T148 I）。与E119 V不同，T148 I在临床样品中未检测到，但在MDCK细胞中的病毒增殖期间获得。使用重组蛋白，T148 I本身显示仅引起扎那米韦50%抑制浓度（IC 50）的6倍增加，并且对其他药物的抑制没有影响。T148 I取代使NA活性降低了50%，最有可能是通过影响NA催化位点处150环的定位。使用焦磷酸测序，在测试的150个MDCK细胞生长的A（H3 N2）病毒中的35个（23%）中检测到T148处的变化，这低于D151处的变化频率（85%），D151是先前与细胞选择有关的NA残基。我们证明，在低感染复数下培养A（H3 N2）病毒（n = 11）可以延迟NA变体的出现，其中第148和/或151位发生变化，尤其是在MDCK-SIAT 1细胞中进行时。我们的研究结果强调了目前在监测甲型H3 N2流感病毒对NAI类抗病毒药物的敏感性方面所面临的挑战。
Assessment of drug susceptibility has become an integral part of influenza virus surveillance. In this study, we describe the drug resistance profile of influenza A(H3N2) virus, A/Mississippi/05/2011, collected from a patient treated with oseltamivir and detected via surveillance. An MDCK cell-grown isolate of this virus exhibited highly reduced inhibition by the neuraminidase (NA) inhibitors (NAIs) oseltamivir (8,005-fold), zanamivir (813-fold), peramivir (116-fold), and laninamivir (257-fold) in the NA inhibition assay. Sequence analysis of its NA gene revealed a known oseltamivir-resistance marker, the glutamic acid-to-valine substitution at position 119 (E119V), and an additional change, threonine to isoleucine at position 148 (T148I). Unlike E119V, T148I was not detected in the clinical sample but acquired during viral propagation in MDCK cells. Using recombinant proteins, T148I by itself was shown to cause only a 6-fold increase in the zanamivir 50% inhibitory concentration (IC50) and had no effect on inhibition by other drugs. The T148I substitution reduced NA activity by 50%, most likely by affecting the positioning of the 150 loop at the NA catalytic site. Using pyrosequencing, changes at T148 were detected in 35 (23%) of 150 MDCK cell-grown A(H3N2) viruses tested, which was lower than the frequency of changes at D151 (85%), an NA residue previously implicated in cell selection. We demonstrate that culturing of the A(H3N2) viruses (n = 11) at a low multiplicity of infection delayed the emergence of the NA variants with changes at position 148 and/or 151, especially when conducted in MDCK-SIAT1 cells. Our findings highlight the current challenges in monitoring susceptibility of influenza A(H3N2) viruses to the NAI class of antiviral drugs.