Collective interactions augment influenza A virus replication in a host-dependent manner

Infection with a single influenza A virus (IAV) is only rarely sufficient to initiate productive infection. Instead, multiple viral genomes are often required within a given cell. Here, we show that IAV reliance 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. In mammals, an additional form of virus-virus interaction is needed. 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 IAV reliance on multiple infection and underscore the importance of virus-virus interactions in IAV infection, evolution and emergence.