Unravelling highly pathogenic influenza virus emergence

揭开高致病性流感病毒出现的谜团

• 批准号: 10718091
• 负责人: Mathilde Richard
• 金额: $ 38.47万
• 依托单位: ERASMUS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10718091
• 关键词: Address; Adenine; Animal Experimentation; Animal Welfare; Animals; Anser species; Automobile Driving; Avian Influenza; Avian Influenza A Virus; Basic Amino Acids; Biological Assay; Birds; Cessation of life; Chicken Cells; Chickens; Coculture Techniques; Code; DNA Resequencing; DNA-Directed RNA Polymerase; Disease; Disease Outbreaks; Domestic Fowls; Ducks; Ecology; Economics; Endothelial Cells; Epithelial Cells; Geese; Generations; Genes; Genetic; Genetic Recombination; Genetic Transcription; Goals; H5 hemagglutinin; H5 influenza virus; H7 hemagglutinin; Health; Hemagglutinin; Human; In Vitro; Industry; Infection; Influenza; Integration Host Factors; Intervention; Knowledge; Laridae; Measures; Modeling; Molecular; Mutation; Natural Selections; North America; Nucleotides; Pathogenicity; Peptide Hydrolases; Polymerase; Prevention; Process; RNA; RNA Sequences; RNA replication; Research; Risk Reduction; Route; Site; Species Specificity; Specificity; Structure; Stuttering; Systemic disease; Systemic infection; Testing; Turkey bird; Uracil; Viral; Viral Genome; Viral Hemagglutinins; Virus; Virus Replication; Zoonoses; airway epithelium; design; experimental study; improved; in vitro Assay; in vitro Model; influenza infection; influenza outbreak; influenzavirus; intestinal epithelium; mortality; new pandemic; novel; pandemic potential; programs; single molecule; spillover event; stem; transmission process; viral RNA

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.

概括。高度致病的鸟类影响病毒（HPAIVS）（“鸟流感”）破坏了家禽行业 野生动植物，损害经济，构成永久性的大流行威胁。 HPAIV来自低致病性 从野生水禽传播（例如鸭子，鹅，海鸥）时 水库，陆地家禽（例如鸡，火鸡）。从LPAIV到HPAIV的过渡是由 插入病毒蛋白酶清洁位点中编码多个碱性氨基酸的核荷兰氏体 黑凝集素（HA）基因在通过影响扎病毒聚合酶复制病毒基因组时。这个变化 在HA中导致全身性病毒传播，其特征是家禽中的内皮动物，死亡率 最多100％。相比之下，HPAIV的全身病毒传播，严重疾病和内皮效果 在大多数鸭，野生和家族的鸭子中，感染很少见或不存在。有趣的是，从LPAIV过渡 仅在H5和H7亚型的影响力病毒中观察到HPAIV。而且，虽然lpaivs 在野生水禽中广泛循环，没有证据表明它们可以演变成这些物种中的HPAIV。 HPAIV的出现目前是不可预测的，因为通过核苷酸初始出现的机制 影响Za病毒聚合酶的插入以及随后在鸟类宿主中的自然选择过程仍然存在 理解不佳。为了了解核插入的分子机制，我们最近预测了 HA裂解位点的亚型特异性RNA茎环结构。在这里，我们假设 病毒RNA复制期间的茎环结构刷新，导致模板闭合，捕获 循环中的聚合酶并导致其口吃和插入核苷酸。此外，我们假设该特定 H5和H7茎环中存在的RNA序列确定了为什么仅在这些亚型中发生插入。到 检验这些假设，我们成功地开发了体外聚合酶测定，包括单分子测定， 通过圆形可以用高吞吐量可靠地检测到HA RNA中的核苷酸插入 重新陈述。其次，我们假设HPAIV系统扩散的能力的内在差异 在家禽与水禽中，确定了从LPAIV的HPAIV选择的过程，并解释了 HPAIV出现的宿主规格特异性。更具体地说，我们假设在 由于它们在家禽中的内皮仿真，而不是在水禽中 传播。为了检验这一假设，我们将LPAIV和HPAIV之间的竞争实验设计为 研究鸡（作为家禽的模型）和鸭子（作为模型的宿主选择的自然选择） 水禽）和在细胞水平上使用新开发的体外Transwell共培养模型 鸡和鸭的呼吸道和肠上皮和内皮细胞。对知识的增加 HPAIV的出现将填补有关影响力的关键知识差距，并可能为 预测并因此可能控制 - HPAIV的出现和随后威胁的爆发 动物和人类健康。