Unravelling highly pathogenic influenza virus emergence

总结。高致病性禽流感病毒(HPAIV)摧毁家禽养殖业，威胁 野生动物，破坏经济，并构成永久的大流行威胁。低致病性HPAIV出现 禽流感病毒(LPAIV)从野生水禽(如鸭、鹅、鸥)传播时，其主要 从水库到陆生家禽(如鸡、火鸡)。从LPAIV到HPAIV的过渡源于 编码多种碱性氨基酸的核苷酸插入病毒的蛋白酶裂解位点 流感病毒聚合酶复制病毒基因组过程中的血凝素(HA)基因。这一变化 在家禽中，HA导致全身性病毒传播，其特征是亲内皮细胞，死亡率高 高达100%。相比之下，全身性病毒传播、严重疾病和HPAIV的内皮亲和性 在大多数种类的野鸭和家鸭中，感染是罕见的或没有的。有趣的是，从LPAIV过渡到 HPAIV仅在H5和H7亚型流感病毒中观察到。此外，尽管LPAIV 虽然在野生水禽中广泛传播，但没有证据表明它们可以在这些物种中进化为HPAIV。 HPAIV的出现目前是不可预测的，因为通过核苷酸的初始出现机制 通过流感病毒聚合酶插入，以及随后在禽类宿主中的自然选择过程仍然存在 人们对此知之甚少。为了了解核苷酸插入的分子机制，我们最近预测 HA裂解部位的亚型特异性RNA茎环结构。在这里，我们假设 在病毒RNA复制过程中，茎环结构重新折叠，导致模板关闭，从而捕获 聚合酶在环路中，导致它结巴并插入核苷酸。此外，我们假设特定的 H5和H7茎环中存在的RNA序列决定了为什么插入只出现在这些亚型中。至 为了验证这些假设，我们成功地开发了体外聚合酶分析，包括单分子分析， 利用该方法，可以通过环路以高通量可靠地检测HA RNA中的核苷酸插入 重新排序。其次，我们假设HPAIV在系统传播能力上的内在差异 在家禽和水禽中确定从LPAIV中自然选择HPAIV的过程并解释 宿主种--HPAIV出现的专一性。更具体地说，我们假设HPAIV是在 家禽而不是水禽，因为它们在家禽中具有促内皮作用，支持其全身 传播。为了验证这一假设，我们设计了LPAIV和HPAIV之间的竞争实验，以 研究鸡(作为家禽的模型)和鸭子(作为模型)在宿主水平上的自然选择 水禽)，并在细胞水平上使用新建立的原代 鸡和鸭的呼吸道和肠道上皮细胞和内皮细胞。增加有关以下方面的知识 HPAIV的出现将填补有关流感的关键知识空白，并可能提供一个行动点 预测--并可能控制--HPAIV的出现和随后具有威胁性的疫情 动物和人类健康。

SUMMARY. Highly pathogenic avian influenza viruses (HPAIVs) (“bird flu”) devastate the poultry industry, threaten wildlife, damage economies, and constitute a permanent pandemic threat. HPAIVs emerge from low pathogenic avian influenza viruses (LPAIVs) upon transmission from wild waterfowl (e.g., ducks, geese, gulls), their main reservoir, to terrestrial poultry (e.g., chickens, turkeys). The transition from LPAIV to HPAIV results from the insertion of nucleotides coding for multiple basic amino acids in the protease cleavage site of the viral hemagglutinin (HA) gene during replication of the viral genome by the influenza virus polymerase. This change in HA leads to systemic virus dissemination characterized by an endotheliotropism in poultry with mortality rates up to 100%. In contrast, systemic virus dissemination, severe disease and endotheliotropism upon HPAIV infection are rare or absent in most species of duck, wild and domestic. Interestingly, the transition from LPAIV to HPAIV has only been observed in influenza viruses of the H5 and H7 subtypes. Moreover, although LPAIVs circulate extensively in wild waterfowl, there is no evidence that they can evolve into HPAIVs in these species. HPAIV emergence is currently unpredictable because the mechanisms of initial emergence through nucleotide insertion by the influenza virus polymerase, and subsequent process of natural selection in avian hosts remain poorly understood. To understand the molecular mechanism of nucleotide insertion, we have recently predicted subtype-specific RNA stem-loop structures at the HA cleavage site. Here, we hypothesize that the stem of the stem-loop structure refolds during viral RNA replication leading to the template closing on itself, trapping the polymerase in the loop and causing it to stutter and insert nucleotides. Additionally, we hypothesize that specific RNA sequences present in H5 and H7 stem-loops determine why insertions only occur in these subtypes. To test these hypotheses, we successfully developed in vitro polymerase assays, including single-molecule assays, with which nucleotide insertions in HA RNA can be reliably detected with high throughput via circular resequencing. Secondly, we hypothesize that intrinsic differences in the ability of HPAIV to spread systemically in poultry versus waterfowl determine the process of natural selection of HPAIVs from LPAIVs and explain the host species-specificity of HPAIV emergence. More specifically, we hypothesize that HPAIV are selected in poultry and not in waterfowl because of their endotheliotropism in poultry supporting their systemic dissemination. To test this hypothesis, we designed competition experiments between LPAIV and HPAIV to study the natural selection at the host level in chickens (as a model for poultry) and ducks (as a model for waterfowl) and at the cellular level using newly developed in vitro transwell co-culture models of primary respiratory and intestinal epithelial and endothelial cells of chickens and ducks. Increased knowledge about HPAIV emergence will fill crucial knowledge gaps on influenza and may provide a point of action to predict – and thus possibly control - HPAIV emergence and subsequent outbreaks that are threatening animal and human health.