Infection with a single influenza A virus (IAV) is only rarely sufficient to initiate productive infection. Instead, multiple viral genomes are often required in a given cell. Here, we show that the reliance of IAV on multiple infection can form an important species barrier. Namely, we find that avian H9N2 viruses representative of those circulating widely at the poultry–human interface exhibit acute dependence on collective interactions in mammalian systems. This need for multiple infection is greatly reduced in the natural host. Quantification of incomplete viral genomes showed that their complementation accounts for the moderate reliance on multiple infection seen in avian cells but not the added reliance seen in mammalian cells. An additional form of virus–virus interaction is needed in mammals. We find that the PA gene segment is a major driver of this phenotype and that both viral replication and transcription are affected. These data indicate that multiple distinct mechanisms underlie the reliance of IAV on multiple infection and underscore the importance of virus–virus interactions in IAV infection, evolution and emergence.

感染单一甲型流感病毒 (IAV) 很少足以引发生产性感染。相反，在一个给定的细胞中通常需要多个病毒基因组。在这里，我们表明 IAV 对多重感染的依赖可以形成一个重要的物种屏障。也就是说，我们发现代表在家禽-人界面广泛传播的禽类 H9N2 病毒表现出对哺乳动物系统中集体相互作用的严重依赖。这种对多重感染的需求在自然宿主中大大减少。对不完整病毒基因组的定量分析表明，它们的互补是对禽类细胞中对多重感染的适度依赖，而不是哺乳动物细胞中对多重感染的依赖。哺乳动物需要另一种形式的病毒-病毒相互作用。我们发现 PA 基因片段是这种表型的主要驱动因素，并且病毒复制和转录都受到影响。这些数据表明，多种不同的机制是 IAV 对多重感染的依赖的基础，并强调了病毒-病毒相互作用在 IAV 感染、进化和出现中的重要性。