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 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. Productive influenza infection can be improved by cooperation and this varies between viral strains and hosts. By quantifying the rates of reassortment and virus production using several methods, including single-cell sequencing, the authors find that isolates of the avian H9N2 influenza subtype are dependent on infections with a second virus, but only in mammalian cells and not in avian cells. These findings are supported by in vivo experiments in guinea pigs and quail. The authors find indications that this type of cooperation between influenza A viruses depends on the RNA polymerase subunit PA.