Emergence and Magnitude of ML336 Resistance in Venezuelan Equine Encephalitis Virus Depend on the Microenvironment.
Emergence and Magnitude of ML336 Resistance in Venezuelan Equine Encephalitis Virus Depend on the Microenvironment.
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DOI：
10.1128/jvi.00317-20
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发表时间：
2020-10-27
影响因子：
5.4
通讯作者：
Jonsson CB
中科院分区：
文献类型：
--
作者：
Lee J;Parvathareddy J;Yang D;Bansal S;O'Connell K;Golden JE;Jonsson CB
RNA病毒，包括委内瑞拉马脑炎病毒（VEEV），具有很高的突变率，能够迅速适应环境中的选择压力。抗病毒化合物在感染期间对病毒种群施加这样的压力。下一代测序允许在种群水平上对病毒进行检查，从而可以跟踪种群中低水平的单核苷酸多态性。因此，可以对出现抗病毒药物耐药性的时间和程度进行跟踪和评估。我们在这里表明，在VEEV中，抗病毒抗性的轨迹和渗透反映了病毒群体复制的微环境。总之，我们展示了在抗病毒压力和两种不同细胞类型下单一种群中VEEV的多样性，并且我们表明可以检查这些病毒的种群动态，以更好地了解它们如何随时间进化。委内瑞拉马脑炎病毒（VEEV）是一种新世界甲病毒，可通过受感染的蚊子传播，在人和马中引起神经系统疾病和死亡。尽管VEEV的持续流行威胁，以及它作为生物恐怖主义制剂的潜在用途，但目前还没有fda批准的抗病毒药物或疫苗用于治疗或预防。在此之前，我们报道了一个小分子ML336的发现，它对VEEV具有有效的抗病毒活性。为了进一步探索ML336的群体水平抗性谱，我们开发了一种全基因组下一代测序（NGS）方法，分别在非人类灵长类动物肾上皮细胞和人类星形胶质细胞细胞系Vero 76和SVGA中进行剂量增加研究，以检测病毒传代的单核苷酸多态性（snp）。我们将VEEV TC-83在这两个细胞系中以7种浓度的ML336传代，从50 nM开始。NGS揭示了非结构蛋白（nsp3）和nsP4基因的几个显著突变，这些突变在这两种不同的体外环境中一致出现——值得注意的是，nsP4的Q210突变。在没有ML336的情况下，这些突变中的一些在Vero 76细胞中传代后是稳定的。网络分析表明，Vero和SVGA的抗性轨迹不同。此外，SNPs在SVGA中的渗透率较低。总之，我们发现微环境影响了VEEV TC-83的SNP谱。了解VEEV对新开发的抗病毒化合物的耐药动力学将指导设计最佳候选药物和给药方案，以最大限度地减少耐药病毒的出现。RNA病毒，包括委内瑞拉马脑炎病毒（VEEV），具有高突变率，可以快速适应环境中的选择压力。抗病毒化合物在感染期间对病毒种群施加这样的压力。下一代测序允许在种群水平上对病毒进行检查，从而可以跟踪种群中低水平的单核苷酸多态性。因此，可以对出现抗病毒药物耐药性的时间和程度进行跟踪和评估。我们在这里表明，在VEEV中，抗病毒抗性的轨迹和渗透反映了病毒群体复制的微环境。总之，我们展示了在抗病毒压力和两种不同细胞类型下单一种群中VEEV的多样性，并且我们表明可以检查这些病毒的种群动态，以更好地了解它们如何随时间进化。
RNA viruses, including Venezuelan equine encephalitis virus (VEEV), have high mutation rates that allow for rapid adaptation to selective pressures in their environment. Antiviral compounds exert one such pressure on virus populations during infections. Next-generation sequencing allows for examination of viruses at the population level, which enables tracking of low levels of single-nucleotide polymorphisms in the population over time. Therefore, the timing and extent of the emergence of resistance to antivirals can be tracked and assessed. We show here that in VEEV, the trajectory and penetration of antiviral resistance reflected the microenvironment in which the virus population replicates. In summary, we show the diversity of VEEV within a single population under antiviral pressure and two distinct cell types, and we show that population dynamics in these viruses can be examined to better understand how they evolve over time. Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus that can cause neurological disease and death in humans and equines following transmission from infected mosquitoes. Despite the continued epidemic threat of VEEV, and its potential use as a bioterrorism agent, there are no FDA-approved antivirals or vaccines for treatment or prevention. Previously, we reported the discovery of a small molecule, ML336, with potent antiviral activity against VEEV. To further explore the population-level resistance profiles of ML336, we developed a whole-genome next-generation sequencing (NGS) approach to examine single nucleotide polymorphisms (SNPs) from virus passaged in dose escalation studies in a nonhuman primate kidney epithelial and a human astrocyte cell line, Vero 76 and SVGA, respectively. We passaged VEEV TC-83 in these two cell lines over seven concentrations of ML336, starting at 50 nM. NGS revealed several prominent mutations in the nonstructural protein (nsP) 3 and nsP4 genes that emerged consistently in these two distinct in vitro environments—notably, a mutation at Q210 in nsP4. Several of these mutations were stable following passaging in the absence of ML336 in Vero 76 cells. Network analyses showed that the trajectory of resistance differed between Vero and SVGA. Moreover, the penetration of SNPs was lower in SVGA. In conclusion, we show that the microenvironment influenced the SNP profile of VEEV TC-83. Understanding the dynamics of resistance in VEEV against newly developed antiviral compounds will guide the design of optimal drug candidates and dosing regimens for minimizing the emergence of resistant viruses. IMPORTANCE RNA viruses, including Venezuelan equine encephalitis virus (VEEV), have high mutation rates that allow for rapid adaptation to selective pressures in their environment. Antiviral compounds exert one such pressure on virus populations during infections. Next-generation sequencing allows for examination of viruses at the population level, which enables tracking of low levels of single-nucleotide polymorphisms in the population over time. Therefore, the timing and extent of the emergence of resistance to antivirals can be tracked and assessed. We show here that in VEEV, the trajectory and penetration of antiviral resistance reflected the microenvironment in which the virus population replicates. In summary, we show the diversity of VEEV within a single population under antiviral pressure and two distinct cell types, and we show that population dynamics in these viruses can be examined to better understand how they evolve over time.