TRANSMISSION OF OSELTAMIVIR-RESISTANT INFLUENZA A H1N1 VIRUSES IN GUINEA PIGS

描述（由申请方提供）：自从奥司他韦（Tamiflu（r））上市以来，偶尔从人体中分离出对奥司他韦具有耐药性的甲型H1 N1和甲型H3 N2流感病毒，通常是从接受药物治疗的患者中分离出[41，43，66-69]。先前的体外和体内数据表明，由于突变的神经氨酸酶（NA）的酶活性受损，耐药病毒的适应性低于敏感菌株[18，48-50，70，71]。然而，季节性甲型H1N1流感病毒对奥司他韦的耐药率突然呈指数级增长--2007年耐药率不到1%，2008年11%，2009年接近100%[51，52] --不能仅仅用奥司他韦使用的选择性压力来解释。这些具有奥司他韦耐药性的甲型H1N1流感病毒除了耐药性外，可能还进化出了比敏感毒株更适合的优势;具体而言，耐药性病毒可能在哺乳动物宿主中更有效地传播。我们实验室的初步数据支持这一假设。对于季节性甲型H1N1流感病毒，旧的模式--即在获得奥司他韦耐药性时，病毒变得不那么适合--可能不再适用。季节性甲型H1N1流感病毒的不同传播性提供了一个及时和相关的案例研究，以探索耐药流感病毒如何进化以克服受损的哺乳动物传播性。 在目前猪源性甲型H1N1流感大流行期间，奥司他韦的使用急剧增加[72];这反过来又增加了在新的甲型H1N1感染者中选择耐药病毒的机会，奥司他韦治疗的个体。事实上，自首次从人体中发现猪源性甲型H1N1流感病毒株以来的7个月内，全世界已检测到25个以上的奥司他韦耐药分离株，主要（但不完全）来自奥司他韦治疗过或未接受奥司他韦治疗的人群[54-56]。迄今为止，所有这些分离株都携带相同的奥司他韦耐药突变-病毒NA残基275处的组氨酸到酪氨酸的变化（NA-H275 Y）-在>99%的季节性A/H1N1病毒中发现。此外，季节性A/H1N1和A/H3 N2病毒继续与猪源性A/H1N1病毒在世界范围内以低水平共同传播[56];理论上，这些毒株之间的基因重配可能产生一种新的人类病毒，具有奥司他韦耐药N1 NA与其他季节性和猪源性基因的组合。利用我们对奥司他韦敏感和耐药的季节性A/H1N1病毒的差异传播性研究中收集的见解，我们建议探索目前的奥司他韦耐药猪源A/H1N1分离株是否比敏感株传播性低，如果是这样，这些病毒如何进化（如季节性A/H1N1病毒），以克服奥司他韦耐药性可能赋予的任何健身缺陷。 1973年，E.D.基尔本写道，“这位研究流感的学生不断回头问‘发生了什么？“希望对过去事件的理解能提醒他注意未来的灾难”[73]; 2009年，我们发现自己处于四十年来的第一次流感大流行之中。我们的目的是，在了解奥司他韦耐药的季节性A/H1N1病毒的最新演变过程中，这些实验不仅可以立即应用于目前的A/H1N1猪源流感大流行，而且还可能产生数据，为我们在未来大流行中的抗病毒预防和治疗提供信息。 项目叙述：2007年，只有不到1%的季节性甲型H1N1流感病毒对口服抗流感药物奥司他韦（达菲）具有耐药性;到2009年，近100%的病毒获得了使其具有耐药性的基因突变。我们假设，这些病毒的进化方式使它们在人类之间变得更容易传播，并且更有效的传播使它们能够如此迅速地变得如此普遍。我们提出的研究旨在了解季节性A/H1N1病毒中发生的遗传变化，以改善哺乳动物传播，并在此过程中评估对奥司他韦耐药的猪源A/H1N1病毒可能沿着类似的路径演变到流行的可能性。

DESCRIPTION (provided by applicant): Since the introduction of oseltamivir (Tamiflu(r)) to the market, oseltamivir-resistant influenza A/H1N1 and A/H3N2 viruses have occasionally been isolated from humans, usually from persons treated with the drug [41, 43, 66-69]. Previous in vitro and in vivo data suggested that drug-resistant viruses were less fit than sensitive strains, due to compromised enzymatic activity of the mutated neuraminidase (NA) [18, 48-50, 70, 71]. However, the sudden, exponential increase in the prevalence of oseltamivir resistance among seasonal A/H1N1 viruses - less than 1% resistant in 2007, 11% resistant in 2008, and near 100% resistant in 2009 [51, 52] - cannot be explained solely by selective pressure of oseltamivir use. These oseltamivir-resistant A/H1N1 viruses may have evolved a fitness advantage over sensitive strains, apart from drug resistance; specifically, resistant viruses may transmit more efficiently among mammalian hosts. Preliminary data from our laboratory supports this hypothesis. For seasonal A/H1N1 virus-es, the old paradigm - that, in acquiring oseltamivir resistance, viruses become less fit - may no longer hold true. The differential transmissibility of seasonal A/H1N1 viruses provides a timely and relevant case study to explore how drug-resistant influenza viruses evolve to overcome impaired mammalian transmissibility. The use of oseltamivir has dramatically increased during the current swine-origin A/H1N1 pandemic [72]; this has in turn increased the chances of selecting resistant viruses in novel A/H1N1-infected, oseltamivir-treated individuals. Indeed, in the seven months since the swine-origin A/H1N1 strain was first recovered from humans, more than 25 oseltamivir-resistant isolates have been detected worldwide, mainly (but not entirely) from oseltamivir-prophylaxed or -treated persons [54-56]. To date, all of these isolates have carried the same oseltamivir resistance mutation - a histidine-to-tyrosine change at residue 275 of the viral NA (NA-H275Y) - that is found in >99% of seasonal A/H1N1 viruses. Furthermore, seasonal A/H1N1 and A/H3N2 viruses have continued to co-circulate at low levels with swine-origin A/H1N1 viruses worldwide [56]; genetic reassortment among these strains could theoretically yield a novel human virus, with an oseltamivir-resistant N1 NA in combination with other seasonal and swine-origin genes. Using insights gleaned from our study of the differential transmissibility of oseltamivir-sensitive and -resistant seasonal A/H1N1 viruses, we propose to explore whether current oseltamivir-resistant swine-origin A/H1N1 isolates are less transmissible than sensitive strains, and, if so, how these viruses might evolve (like seasonal A/H1N1 viruses) to overcome any fitness deficiency that oseltamivir-resistance might confer. In 1973, E.D. Kilbourne wrote that the "student of influenza is constantly looking back over his shoulder and asking 'what happened?' in the hope that understanding of past events will alert him to the catastrophes of the future" [73]; in 2009, we find ourselves amidst the first influenza pandemic in forty years. It is our intention that, in understanding the recent evolution of oseltamivir-resistant seasonal A/H1N1 viruses, these experiments will not only be immediately applicable to the current A/H1N1 swine-origin influenza pandemic, but also might yield data that could inform our approach to antiviral prophylaxis and treatment in future pandemics. Project Narrative: In 2007, less than 1% of seasonal influenza A/H1N1 viruses were resistant to the oral anti-influenza drug oseltamivir (Tamiflu(r)); by 2009, nearly 100% had acquired a genetic mutation that rendered them drug resistant. We hypothesize that these viruses evolved in such a way that they became more transmissible among humans, and that it was more efficient transmission that allowed them to become so prevalent so quickly. The research we propose seeks to understand the genetic changes that occurred in seasonal A/H1N1 viruses to improve mammalian transmission, and in so doing, to assess the likelihood that oseltamivir-resistant swine-origin A/H1N1 viruses might evolve along a similar path to prevalence.