Different genetic determinants for high virulence, transmission and replication of high pathogenicity H7N7 avian influenza virus in turkeys and chickens

High pathogenicity (HP) avian influenza viruses (AIV) generally evolve from low pathogenicity (LP) precursors after transmission from wild birds to chickens (Gallus gallus domesticus) and turkeys (Meleagris gallopavo), causing severe economic losses worldwide. Turkeys are more susceptible to AIV infection than chickens and are considered potential bridging hosts that facilitate the emergence of HPAIV. Beyond the polybasic cleavage site (pCS) in hemagglutinin (HA), little is known about other virulence determinants of HPAIV in these species. In 2015, HPAIV H7N7 and its LP ancestor were isolated from the same chicken farm, which differed by 16 nonsynonymous mutations across all eight gene segments, in addition to the pCS. Here we identify the genetic determinants, including the pCS, that contributed to the HPAIV H7N7 virulence, transmission, replication, and tissue distribution in chickens and turkeys. Notably, the non-structural (NS1) or matrix (M) proteins encoding segments in turkeys, or NS segment in chickens, rendered viruses as virulent and transmissible as the original HPAIV. Endotheliotropism, observed exclusively in chickens, was driven by the pCS and, to a lesser extent, the neuraminidase (NA). In vitro, the M2-V68L mutation influenced NS1 expression and virus morphology in chicken and turkey cells. Additionally, HPAIV NS1 enhanced polymerase activity and effectively suppressed interferon induction, a process further modulated by M2-V68L. These findings underscore the critical role of turkeys as a "hub" in the evolution of HPAIV from LP precursors, offering crucial insights into the genotypic and phenotypic factors that facilitate viral adaptation in different poultry species. ImportanceHigh pathogenicity avian influenza viruses (HPAIV) cause severe economic losses for the poultry industry worldwide. HPAIV generally evolve from low pathogenicity (LP) ancestors in galliform birds, with turkeys being more susceptible to severe disease and death than chickens. The mechanisms underlying HPAIV emergence in these species remain unclear. This study reveals two distinct evolutionary pathways for HPAIV. In turkeys, both the polybasic hemagglutinin cleavage site (pCS) and mutations in the NS or M segments contributed to high virulence and transmission. In chickens, only the NS segment was critical, in addition to the pCS. These segments increased virus replication in both chicken and turkey cells. However, unlike chicken cells, the M and NS segments did not play a role in blocking the innate immune response. Understanding these species-specific mechanisms highlights the role of turkeys as a bridging host and provides insights into the molecular evolution of HPAIV from LP precursors.