Natural adaptation of an antigenically novel avian influenza A virus (IAV) to be transmitted efficiently in humans has the potential to trigger a devastating pandemic. Understanding viral genetic determinants underlying adaptation is therefore critical for pandemic preparedness, as the knowledge gained enhances surveillance and eradication efforts, prepandemic vaccine design, and efficacy assessment of antivirals. However, this work has risks, as making gain-of-function substitutions in fully infectious IAVs may create a pathogen with pandemic potential. Thus, such experiments must be tightly controlled through physical and biological risk mitigation strategies. Here, we applied a previously described biological containment system for IAVs to a 2009 pandemic H1N1 strain and a highly pathogenic H5N1 strain. The system relies on deletion of the essential viral hemagglutinin (HA) gene, which is instead provided in trans, thereby restricting multicycle virus replication to genetically modified HA-complementing cells. In place of HA, a Renilla luciferase gene is inserted within the viral genome, and a live-cell luciferase substrate allows real-time quantitative monitoring of viral replication kinetics with a high dynamic range. We demonstrate that biologically contained IAV-like particles exhibit wild-type sensitivities to approved antivirals, including oseltamivir, zanamivir, and baloxavir. Furthermore, the inability of these IAV-like particles to genetically acquire the host-encoded HA allowed us to introduce gain-of-function substitutions in the H5 HA gene that promote mammalian transmissibility. Biologically contained “transmissible” H5N1 IAV-like particles exhibited wild-type sensitivities to approved antivirals, to the fusion inhibitor S20, and to neutralization by existing H5 monoclonal and polyclonal sera. This work represents a proof of principle that biologically contained IAV systems can be used to safely conduct selected gain-of-function experiments.

中文翻译：

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应用生物学上包含的报告系统来研究具有大流行潜力的功能获得性 H5N1 甲型流感病毒。

具有抗原性的新型甲型禽流感病毒 (IAV) 在人类中有效传播的自然适应有可能引发毁灭性的大流行。因此，了解潜在适应的病毒遗传决定因素对于大流行防范至关重要，因为所获得的知识可以加强监测和根除工作、大流行前疫苗设计以及抗病毒药物的功效评估。然而，这项工作有风险，因为在完全传染性的 IAV 中进行功能获得性替代可能会产生具有大流行潜力的病原体。因此，必须通过物理和生物风险缓解策略严格控制此类实验。在这里，我们将先前描述的 IAV 生物遏制系统应用于 2009 年大流行的 H1N1 毒株和高致病性 H5N1 毒株。trans，从而将多周期病毒复制限制在转基因 HA 补充细胞中。代替 HA，海肾荧光素酶基因插入病毒基因组中，活细胞荧光素酶底物允许以高动态范围实时定量监测病毒复制动力学。我们证明了生物学上包含的 IAV 样颗粒对批准的抗病毒药物表现出野生型敏感性，包括奥司他韦、扎那米韦和巴洛沙韦。此外，这些 IAV 样颗粒无法通过遗传获得宿主编码的 HA，这使我们能够在 H5 HA 基因中引入功能获得性替代，以促进哺乳动物的传播。生物学上包含的“可传播”H5N1 IAV 样颗粒对批准的抗病毒药物、融合抑制剂 S20 以及现有 H5 单克隆和多克隆血清的中和作用表现出野生型敏感性。