College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Republic of Korea.
College of Veterinary Medicine and Agricultural Sciences, De La Salle Araneta University, Potrero Malabon City, Philippines.
J Virol. 2019 Mar 5;93(6). doi: 10.1128/JVI.01825-18. Print 2019 Mar 15.
Neuraminidase (NA) inhibitors (NAIs) are widely used antiviral drugs for the treatment of humans with influenza virus infections. There have been widespread reports of NAI resistance among seasonal A(H1N1) viruses, and most have been identified in oseltamivir-exposed patients or those treated with other NAIs. Thus, monitoring and identifying NA markers conferring resistance to NAIs-particularly newly introduced treatments-are critical to the management of viral infections. Therefore, we screened and identified substitutions conferring resistance to laninamivir by enriching random mutations in the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus followed by deep sequencing of the laninamivir-selected variants. After the generation of single mutants possessing each identified mutation, two A(H1N1)pdm09 recombinants possessing novel NA gene substitutions (i.e., D199E and P458T) were shown to exhibit resistance to more than one NAI. Of note, mutants possessing P458T-which is located outside of the catalytic or framework residue of the NA active site-exhibited highly reduced inhibition by all four approved NAIs. Using MDCK cells, we observed that the viral replication of the two recombinants was lower than that of the wild type (WT). Additionally, in infected mice, decreased mortality and/or mean lung viral titers were observed in mutants compared with the WT. Reverse mutations to the WT were observed in lung homogenate samples from D199E-infected mice after 3 serial passages. Overall, the novel NA substitutions identified could possibly emerge in influenza A(H1N1)pdm09 viruses during laninamivir therapy and the viruses could have altered NAI susceptibility, but the compromised viral fitness may limit viral spreading. With the widespread emergence of NAI-resistant influenza virus strains, continuous monitoring of mutations that confer antiviral resistance is needed. Laninamivir is the most recently approved NAI in several countries; few data exist related to the selection of viral mutations conferring resistance to laninamivir. Thus, we screened and identified substitutions conferring resistance to laninamivir by random mutagenesis of the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus strain followed by deep sequencing of the laninamivir-selected variants. We found several novel substitutions in NA (D199E and P458T) in an A(H1N1)pdm09 background which conferred resistance to NAIs and which had an impact on viral fitness. Our study highlights the importance of continued surveillance for potential antiviral-resistant variants and the development of alternative therapeutics.
神经氨酸酶（NA）抑制剂（NAIs）被广泛用于治疗人类流感病毒感染。季节性 A（H1N1）病毒对 NAI 的耐药性已有广泛报道，且大多数耐药性发生在奥司他韦暴露的患者或接受其他 NAI 治疗的患者中。因此，监测和识别对 NAI 具有耐药性的 NA 标志物——尤其是新引入的治疗方法——对于病毒感染的管理至关重要。因此，我们通过在 2009 年大流行流感 [A（H1N1）pdm09] 病毒的 NA 基因中富集随机突变，然后对筛选出的使用 laninamivir 的变体进行深度测序，筛选和鉴定对 laninamivir 具有耐药性的突变。在生成每个鉴定的突变的单突变体后，两个具有新型 NA 基因取代（即 D199E 和 P458T）的 A（H1N1）pdm09 重组体被证明对一种以上的 NAI 具有耐药性。值得注意的是，位于 NA 活性位点的催化或框架残基之外的 P458T 突变体对所有四种批准的 NAI 的抑制作用均显著降低。使用 MDCK 细胞，我们观察到两种重组体的病毒复制低于野生型（WT）。此外，在感染小鼠中，与 WT 相比，突变体的死亡率和/或肺部病毒滴度降低。在 D199E 感染小鼠的肺匀浆样本中，在连续 3 次传代后观察到向 WT 的反向突变。总体而言，在 laninamivir 治疗期间，新鉴定的 NA 取代可能会出现在甲型流感 [H1N1]pdm09 病毒中，并且病毒可能具有改变的 NAI 敏感性，但病毒适应性的降低可能会限制病毒的传播。随着具有抗药性的流感病毒株的广泛出现，需要持续监测赋予抗病毒抗性的突变。在几个国家，laninamivir 是最近批准的 NAI；关于赋予 laninamivir 耐药性的病毒突变的选择，数据很少。因此，我们通过对 2009 年大流行流感 [A（H1N1）pdm09] 病毒株的 NA 基因进行随机诱变，然后对筛选出的使用 laninamivir 的变体进行深度测序，筛选和鉴定对 laninamivir 具有耐药性的突变体。我们在 A（H1N1）pdm09 背景下发现了几种新型的 NA 取代（D199E 和 P458T），它们赋予了对 NAI 的耐药性，并对病毒适应性产生了影响。我们的研究强调了持续监测潜在的抗病毒耐药变异体和开发替代治疗方法的重要性。
