Influenza A viruses (IAV) cause respiratory disease in many host species, including humans and pigs. The spillover of IAV between swine and humans poses a challenge for the swine industry and public health. IAV have a segmented genome which consists of 8 negative-sense RNA gene segments that encode 10 major proteins and several strain-specific minor proteins. Hemagglutinin (HA), the major surface glycoprotein, is a determining factor for host specificity. IAVs carrying a triple reassortant internal gene (TRIG) constellation, containing genes from human-, swine- and avian-origin, emerged in swine in North America in the late 90s, and has contributed to the establishment of human-origin IAVs in pigs and increased viral diversity. While there is frequent evidence of human IAVs being introduced into swine populations, the molecular determinants driving the adaptation to pigs is not well understood. By using a reassortant recombinant IAV containing surface genes of human-origin IAV, we showed the virus was able to replicate and transmit in pigs. Next, we studied the viral evolution by performing serial passages using differentiated swine cells as an in vitro infection model. Sequencing and variant analysis from the in vivo and in vitro studies revealed several mutations arose after replication in pigs or swine cells. All mutations were on the head of the HA. We reconstructed the mutant viruses via reverse genetics and showed they replicate more efficiently in swine cells compared to viruses containing the wild type human-origin IAV HA. Overall, our results show mutations quickly arise after replication of human-origin IAV HA in swine and result in improved fitness.

INDEX WORDS: Influenza A virus, Hemagglutinin, Viral evolution, Host specificity, Triple reassortant internal gene (TRIG), Next generation sequencing, Reverse Genetics, Variant Analysis, Viral growth kinetics, Primary swine epithelial cell, Cell differentiation, Serial passage