The generation and application of replication-competent influenza A virus (IAV) expressing a reporter gene represent a valuable tool to elucidate the mechanism of viral pathogenesis and establish new countermeasures to combat the threat of influenza. Here, replication-competent IAVs with a neuraminidase (NA) segment harboring a fluorescent reporter protein, Venus, were generated in the background of H5N1, H7N9, and H9N2 influenza viruses, the three subtypes of viruses with imminent pandemic potential. All three reporter viruses maintained virion morphology, replicated with similar or slightly reduced titers relative to their parental viruses, and stably expressed the fluorescent signal for at least two passages in embryonated chicken eggs. As a proof of concept, we demonstrated that these reporter viruses, used in combination with a high-content imaging system, can serve as a convenient and rapid tool for the screening of antivirals and host factors involved in the virus life cycle. Moreover, the reporter viruses demonstrated similar growth properties and tissue tropism as their parental viruses in mice, among which the H7N9 NA-Venus virus could potentially be used in ex vivo studies to better understand H7N9 pathogenesis or to develop novel therapeutics.

表达报告基因的具有复制能力的甲型流感病毒 (IAV) 的产生和应用是阐明病毒发病机制和建立新对策以应对流感威胁的宝贵工具。在这里，在 H5N1、H7N9 和 H9N2 流感病毒的背景下产生了具有复制能力的 IAVs，其具有含有荧光报告蛋白 Venus 的神经氨酸酶 (NA) 片段，这三种病毒具有迫在眉睫的大流行潜力。所有三种报告病毒都保持病毒体形态，复制时的滴度与其亲本病毒相似或略有降低，并且在含胚鸡蛋中至少两次传代时稳定表达荧光信号。作为概念证明，我们证明了这些报告病毒，与高内涵成像系统结合使用，可以作为筛选病毒生命周期中涉及的抗病毒药物和宿主因素的方便快捷的工具。此外，报告病毒在小鼠体内表现出与其亲本病毒相似的生长特性和组织嗜性，其中 H7N9 NA-Venus 病毒有可能用于离体研究，以更好地了解 H7N9 发病机制或开发新疗法。