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

甲型流感病毒与细胞表面的唾液酸结合，从而进入细胞内。基因组复制后，新的病毒粒子通过宿主细胞膜存在。神经氨酸酶(NA)是一种包膜表面糖蛋白，通过裂解唾液酸促进感染颗粒的最终释放和扩散。已知的NA亚型有12种。神经氨酸酶抑制剂(NAI)，如达菲，是重要的抗病毒药物，NAI与NA活性部位的唾液酸竞争结合，防止病毒复制。这些药物用于人类预防和治疗流感感染，无论是在季节性流行期间还是在大流行情况下。对NAI产生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有所不同。大多数的功能性耐药性图谱已经在人类流感病毒株n1和n2亚型中进行了，而关于禽流感nA亚型的需求的信息只有少量报道。由于20世纪的三次人类流感大流行都是由流感毒株引起的，其中NA基因来自禽源，而且仍有许多不同亚型的禽流感病毒频繁发生人畜共患感染，因此了解已知的NAI相关基序是否对禽流感NA亚型具有抵抗力以及是否存在其他未知的抗药性基序似乎是至关重要的，这些应作为禽类监测计划的一部分。因此，我们将在威胁人类健康的禽流感病毒的NA中引入特定的突变，并解决它们是否会对当前的NAI药物和正在开发的药物产生抗药性。我们还将尝试确定NA中是否有任何新的突变可以导致禽亚型的抗药性。20世纪90年代中期，作用于流感M2蛋白的抗流感病毒药物金刚烷胺在家禽中的无管制使用导致了全球禽流感的广泛耐药性。这将可用于治疗人类人畜共患流感感染的武器库减少到只有NAI。在禽流感病毒传播的持续威胁下，NAI药物很可能会在家禽中不受监管地使用，除非这一次加强监管。病毒学家不清楚的是，对NAI药物具有抗药性的禽流感病毒是否会在家禽宿主中具有竞争力，并在传播后保持稳定。我们将在这项建议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抗药性突变，并在鸡只中测试它们感染和传播病毒的能力。主要关注的是下一次人类流感大流行可能对NAI产生抗药性。可能出现这种情况的情况是，首先将一种已经具有NAI耐药性的禽类菌株直接引入人类群体。其次，由于靶受体和粘液环境的改变，将禽类毒株跨物种引入新的宿主环境(人或家禽中的猪)可能会导致病毒表面蛋白的唾液酸结合和切割活性的失衡。面对NAI用于治疗和预防的热情，不平衡的活动可能会导致NAI耐药基序得到更好的支持和更有可能发展。因此，在这项工作计划中，我们将用禽流感病毒株感染模仿鸟类、猪和人类宿主的生物组织底物，并应用NAI药物奥司他韦。使用下一代测序，我们可以确定NAS对重要禽流感毒株如H9N2和H7N9的NAI耐药性的发展速度，以及不同宿主环境对耐药性产生的可能性的影响。

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.