Unravelling highly pathogenic influenza virus emergence

摘要高致病性禽流感病毒（HPAIV）（“禽流感”）危害家禽业，威胁 野生动物，破坏经济，并构成永久性的流行病威胁。HPAIV出现于低致病性 禽流感病毒（LPAIV）在从野生水禽传播时（例如，鸭、鹅、海鸥），它们的主要 水库，陆生家禽（例如，鸡、火鸡）。从LPAIV到HPAIV的转变是由于 在病毒的蛋白酶切割位点插入编码多个碱性氨基酸的核苷酸 在流感病毒聚合酶复制病毒基因组的过程中，血凝素（HA）基因被激活。这种变化 HA导致家禽中以向内皮细胞性为特征的系统性病毒传播，并导致死亡率 高达100%。相反，HPAIV引起的全身性病毒传播、严重疾病和内皮细胞嗜性 在大多数野生和家养鸭种中，感染是罕见或不存在的。有趣的是，从LPAIV 仅在H5和H7亚型的流感病毒中观察到HPAIV。此外，虽然LPAIV 虽然它们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成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的机制， 流感病毒聚合酶的插入以及随后在禽类宿主中的自然选择过程仍然存在 不太了解。为了了解核苷酸插入的分子机制，我们最近预测 HA切割位点的亚型特异性RNA茎环结构。在这里，我们假设， 茎环结构在病毒RNA复制过程中重新折叠，导致模板自身闭合，捕获病毒RNA。 环中的聚合酶，并导致它口吃和插入核苷酸。另外，我们假设， 存在于H5和H7茎环中的RNA序列决定了为什么插入只发生在这些亚型中。到 为了验证这些假设，我们成功地开发了体外聚合酶测定，包括单分子测定， 利用该方法，可以通过环状寡核苷酸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系统传播能力的内在差异 在家禽和水禽中，确定了HPAIV从LPAIV中自然选择的过程，并解释了 HPAIV出现的宿主种特异性。更具体地说，我们假设HPAIV被选择在 家禽而不是水禽，因为它们在家禽中的亲内皮性支持它们的系统性 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， 研究鸡（作为家禽的模型）和鸭（作为 水禽）和在细胞水平使用新开发的体外transwell共培养模型的原代 呼吸道和肠道上皮细胞和内皮细胞。增加了关于 HPAIV的出现将填补流感方面的关键知识空白，并可能提供一个行动点， 预测-从而可能控制- HPAIV的出现和随后的爆发， 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.