PB1-F2 protein, the 11th influenza A virus (IAV) protein, is considered to play an important role in primary influenza virus infection and postinfluenza secondary bacterial pneumonia in mice. The functional role of PB1-F2 has been reported to be a strain-specific and host-specific phenomenon. Its precise contribution to the pathogenicity and transmission of influenza virus in mammalian host, such as swine, and avian hosts, such as turkeys, remain largely unknown. In this study, we explored the role of PB1-F2 protein of triple-reassortant (TR) H3N2 swine influenza virus (SIV) in pigs and turkeys. Using the eight-plasmid reverse genetics system, we rescued wild-type SIV A/swine/Minnesota/1145/2007 (H3N2) (SIV 1145-WT), a PB1-F2 knockout mutant (SIV 1145-KO), and its N66S variant (SIV 1145-N66S). The ablation of PB1-F2 in SIV 1145 modulated early-stage apoptosis but did not affect the viral replication in swine alveolar macrophage cells. In pigs, PB1-F2 expression did not affect nasal shedding, lung viral load, immunophenotypes, and lung pathology. On the other hand, in turkeys, SIV 1145-KO infected poults, and its in-contacts developed clinical signs earlier than SIV 1145-WT groups and also displayed more extensive histopathological changes in intestine. Further, turkeys infected with SIV 1145-N66S displayed poor infectivity and transmissibility. The more extensive histopathologic changes in intestine and relative transmission advantage observed in turkeys infected with SIV 1145-KO need to be further explored. Taken together, these results emphasize the host-specific roles of PB1-F2 in the pathogenicity and transmission of IAV.

IMPORTANCE Novel triple-reassortant H3N2 swine influenza virus emerged in 1998 and spread rapidly among the North American swine population. Subsequently, it showed an increased propensity to reassort, generating a range of reassortants. Unlike classical swine influenza virus, TR SIV produces a full-length PB1-F2 protein, which is considered an important virulence marker of IAV pathogenicity. Our study demonstrated that the expression of PB1-F2 does not impact the pathogenicity of TR H3N2 SIV in pigs. On the other hand, deletion of PB1-F2 caused TR H3N2 SIV to induce clinical disease early and resulted in effective transmission among the turkey poults. Our study emphasizes the continuing need to better understand the virulence determinants for IAV in intermediate hosts, such as swine and turkeys, and highlights the host-specific role of PB1-F2 protein.