The development of neuraminidase inhibitor resistance in avian influenza: genetic determinants, fitness cost and zoonotic transmission.

禽流感中神经氨酸酶抑制剂耐药性的发展：遗传决定因素、适应成本和人畜共患传播。

• 批准号: BB/M023362/1
• 负责人: Holly Shelton
• 金额: $ 48.63万
• 依托单位: The Pirbright Institute
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM023362%2F1
• 关键词: development; neuraminidase; inhibitor; resistance; avian

Influenza A viruses bind sialic acid on the cell surface allowing entry. After genome replication, new virions exit through host cell membranes. Neuraminidase (NA) is present as an envelope surface glycoprotein and facilitates the final release and spread of infectious particles by cleaving sialic acid. There are 12 known NA subtypes. Neuraminidase inhibitors (NAI), such as Tamiflu are important antivirals, where the NAIs compete for binding with sialic acid in the active site of NA preventing viral replication. These drugs are utilised for humans prophylactically and for treatment upon influenza infection, both during seasonal epidemics and in pandemic scenarios. The exact amino acid mutations that confer resistance to NAIs differ between NA subtypes and the drug to which the resistance is directed. Most of the functional resistance mapping has been undertaken in NAs of human influenza strains, the N1 and N2 subtypes, whilst only a small amount of information has been reported about the requirements in avian NA subtypes. Since the three major human influenza pandemics of the 20th Century were caused by influenza strains where the NA gene originated from an avian source and there continues to be frequent zoonotic infections from avian influenza viruses of many different subtypes, it seems critical to understand whether known NAI associated motifs confer resistance to avian NA subtypes and whether other unknown motifs for resistance exist that should be part of avian surveillance programmes. We will therefore introduce specific mutations in the NA of avian influenza viruses that threaten human health and address whether they confer a resistant profile to the current NAI drugs and those in development. We will also attempt to identify if any novel mutations in NA can cause resistance in avian subtypes. In the mid-1990s the unregulated use of the influenza anti-viral Amantadine, which acts on the M2 protein of influenza, in poultry resulted in widespread resistance in avian influenza globally. This reduced the arsenal available to treat zoonotic influenza infection in humans to only the NAIs. Under continued threat from circulating avian influenza viruses the same unregulated use in poultry is likely with NAI drugs unless the regulation is more strongly enforced this time around. What virologists are unclear about is whether avian influenza viruses that are resistant to NAI drugs would be competitive in the poultry host and be stably maintained upon transmission. These are question we will endeavour to answer in this proposal by introducing the most prevalent NAI resistance mutations in to important avian influenza NA subtypes and measuring in chickens their ability to infect and transmit the viruses.Of major concern is the potential for the next human influenza pandemic to harbour or rapidly acquire resistance to NAIs. The scenarios by which this could manifest are firstly by the direct introduction to the human population of an avian strain that already carries NAI resistance. Secondly, the cross-species introduction of an avian strain in to a new host environment (humans or swine from poultry) may result in the unbalancing of sialic acid binding and cleavage activity of the virus surface proteins due to a change in the target receptors and mucus environment. An unbalanced activity in the face of enthusiastic NAI use for treatment and prophylaxis, may result in NAI resistance motifs being better supported and more likely to develop. Therefore in this programme of work we will infect biological tissue substrates that mimic avian, swine and human hosts with avian influenza strains and apply the NAI drug Oseltamivir. Using next generation sequencing we can determine the rate of NAI resistance development in the NAs from important avian influenza strains such as H9N2 and H7N9 and the effect that the different host environments have on the likelihood that resistance will develop.

甲型流感病毒结合细胞表面上的唾液酸，从而允许进入。基因组复制后，新的病毒体通过宿主细胞膜离开。神经氨酸酶（NA）作为包膜表面糖蛋白存在，并通过裂解唾液酸促进感染性颗粒的最终释放和扩散。有12种已知的NA亚型。神经氨酸酶抑制剂（NAI），如达菲是重要的抗病毒药，其中NAI竞争与NA的活性位点中的唾液酸结合，防止病毒复制。这些药物在季节性流行和大流行情况下用于人类预防和治疗流感感染。赋予NAI耐药性的确切氨基酸突变在NA亚型和耐药性所针对的药物之间不同。大多数功能性耐药图谱已在人流感毒株N1和N2亚型的NA中进行，而关于禽流感NA亚型的要求仅报告了少量信息。由于世纪的三次主要人类流感大流行是由NA基因源自禽类来源的流感毒株引起的，并且许多不同亚型的禽流感病毒继续频繁地引起人畜共患感染，似乎关键在于了解已知的NAI相关基序是否赋予对禽NA亚型的抗性，以及是否存在其他未知的抗性基序，这些基序应该是禽NA亚型的一部分。监测方案。因此，我们将在威胁人类健康的禽流感病毒NA中引入特定突变，并解决它们是否对当前的NAI药物和正在开发的药物产生耐药性。我们还将尝试确定NA中的任何新突变是否会导致禽类亚型的耐药性。在20世纪90年代中期，在家禽中不受管制地使用流感抗病毒金刚烷胺，其作用于流感的M2蛋白，导致全球禽流感的广泛耐药性。这使治疗人畜共患流感的药物库减少到只有NAI。在禽流感病毒传播的持续威胁下，NAI药物可能会在家禽中同样不受管制地使用，除非这次更严格地执行该法规。病毒学家不清楚的是，对NAI药物具有耐药性的禽流感病毒是否会在家禽宿主中具有竞争力，并在传播时稳定地维持。我们将在本提案中努力回答这些问题，方法是在重要的禽流感NA亚型中引入最普遍的NAI耐药突变，并测量鸡感染和传播病毒的能力。这种情况可能首先是通过直接向人类引入已经携带NAI抗性的禽类菌株。其次，将禽类毒株跨物种引入新的宿主环境（人或来自家禽的猪）可能会导致唾液酸结合和病毒表面蛋白裂解活性的不平衡，这是由于靶受体和粘液环境的变化。面对热情的NAI用于治疗和预防，不平衡的活动可能导致NAI抗性基序得到更好的支持，更有可能发展。因此，在本工作方案中，我们将用禽流感病毒株感染模拟禽类、猪和人类宿主的生物组织基质，并应用NAI药物奥司他韦。使用下一代测序，我们可以确定重要禽流感病毒株（如H9N2和H7N9）的NAs中NAI耐药性的发展速度，以及不同宿主环境对耐药性发展可能性的影响。

项目成果

Functional neuraminidase inhibitor resistance motifs in avian influenza A(H5Nx) viruses.

• DOI: 10.1016/j.antiviral.2020.104886
• 发表时间: 2020-10
• 期刊: Antiviral research
• 影响因子: 7.6
• 作者: Bialy D;Shelton H
• 通讯作者: Shelton H

Defining functional neuraminidase inhibitor drug resistance motifs in avian influenza viruses and the consequential impact on virus fitness in chicken cells

• DOI: 10.1101/663872
• 发表时间: 2019-06
• 期刊: bioRxiv
• 作者: D. Bialy;Holly Shelton