Unravelling highly pathogenic influenza virus emergence

揭开高致病性流感病毒出现的谜团

基本信息

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
项目类别：
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

项目摘要

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.

摘要：高致病性禽流感病毒（HPAIV）（“禽流感”）摧毁了家禽业，威胁着家禽业。低致病性高致病性禽流感病毒对野生动物造成损害，并构成永久性的大流行威胁。禽流感病毒 (LPAIV) 通过野生水禽（例如鸭、鹅、海鸥）传播，其主要传播途径是从储存库到陆生家禽（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血凝素（HA）基因在流感病毒聚合酶复制病毒基因组过程中发生这种变化。 HA 导致全身性病毒传播，其特征为家禽中的内皮细胞趋向性，并导致死亡率相比之下，HPAIV 的全身性病毒传播、严重疾病和内皮细胞趋向性。在大多数鸭子品种中，无论是野生还是家养，感染都很罕见或不存在。然而，尽管 LPAIV 只在 H5 和 H7 亚型流感病毒中观察到了 HPAIV。广泛存在于野生水禽中，但没有证据表明它们可以在这些物种中进化为高致病性禽流感病毒。 HPAIV 的出现目前无法预测，因为最初出现的机制是通过核苷酸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为了了解核苷酸插入的分子机制，我们最近进行了预测。 HA 切割位点的亚型特异性 RNA 茎环结构在这里，我们寻找到了 HA 切割位点的茎环结构。病毒RNA复制过程中茎环结构重新折叠，导致模板自身闭合，捕获环中的聚合酶并使其断断续续地插入核苷酸。 H5 和 H7 茎环中存在的 RNA 序列决定了插入仅发生在这些亚型中的原因。为了测试这些假设，我们成功开发了体外聚合酶测定，包括单分子测定，通过循环，可以以高通量可靠地检测 HA RNA 中的核苷酸插入其次，我们追踪 HPAIV 系统传播能力的内在差异。在家禽与水禽中确定 HPAIV 从 LPAIV 中自然选择的过程，并解释 HPAIV 出现的宿主物种特异性更具体地说，我们发现 HPAIV 在以下情况下被选择。家禽而不是水禽，因为它们在家禽中具有内皮促性，支持其全身性为了检验这一假设，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（作为家禽的模型）在宿主水平上的自然选择水禽）并在细胞水平上使用新开发的原代细胞体外 Transwell 共培养模型增加了对鸡和鸭呼吸道和肠道上皮细胞和内皮细胞的了解。 HPAIV 的出现将填补流感方面的关键知识空白，并可能为以下方面提供行动要点：预测 — 从而可能控制 — HPAIV 的出现和随后的威胁性爆发动物和人类健康。