Transmission of Human Influenza A Virus in Pigs Selects for Adaptive Mutations on the HA Gene

Influenza A viruses (IAV) cause respiratory diseases in many host species, including humans and pigs. The spillover of IAV between swine and humans has been a concern for both public health and the swine industry. With the emergence of the triple reassortant internal gene (TRIG) constellation, establishment of human-origin IAVs in pigs has become more common, leading to increased viral diversity. However, little is known about the adaptation processes that are needed for a human-origin IAV to transmit and become established in pigs. We generated a reassortant IAV containing surface gene segments from a human IAV strain and internal gene segments from the 2009 pandemic and TRIG IAV lineages and demonstrated that it can replicate and transmit in pigs. Sequencing and variant calling analysis identified a mutant that emerged during replication in pigs, which was mapped to a region near the receptor binding site of the hemagglutinin (HA). The variant was present in all contact pigs and replicated more efficiently in differentiated swine tracheal cells compared to the virus containing the wildtype human-origin HA. These results show that variants are selected quickly after replication of human-origin HA in pigs, leading to improved fitness in the swine host, likely contributing to transmission. Importance Influenza A viruses (IAV) cause respiratory disease in several species, including humans and pigs. The bidirectional transmission of IAV between humans and pigs plays a significant role in the generation of novel viral strains, greatly impacting viral epidemiology. However, little is known about the evolutionary processes that allow human IAV to become established in pigs. In this study, we generated reassortant viruses containing human seasonal HA and NA on different constellations of internal genes and tested their ability to replicate and transmit in pigs. We demonstrated that a virus containing a common internal gene constellation currently found in U.S. swine was able to transmit efficiently via the respiratory route. We identified a specific amino acid mutation that was fixed in the respiratory contact pigs that was associated with improved replication in primary swine tracheal epithelial cells, suggesting it was crucial for the transmissibility of the human virus in pigs.