The mechanisms and consequences of evolution on viral fitness following interspecies transmission and long-term adaptation are poorly understood. Influenza A viruses (IAVs) circulate among wild birds and have been linked to the emergence of viruses of humans, pigs, dogs, and horses. Since the mid 1950s, equine influenza viruses (EIVs) have emerged in Europe (H7N7, in 1956), America (H3N8, in 1963) and Asia (H3N8, in 1989). All EIVs are thought to have originated in birds, and only the H3N8 EIV lineage that emerged in 1963 circulates today. I used EIVs to study changes in viral fitness following host shifts. To this end, I compared the in vitro and ex vivo infection phenotypes of A/equine/Lexington/1/1966 (EIV/66), a representative of the extinct H7N7 EIV lineage; A/equine/Jilin/1/1989 (EIV/89), also extinct; and two viruses of the currently circulating H3N8 EIV lineage: A/equine/Uruguay/1/1963 (EIV/63) and its descendant A/equine/Ohio/1/2003 (EIV/2003). I also studied A/ruddy shelduck/Mongolia/963v/2009 (AIV/2009), an avian influenza virus phylogenetically related to EIV/89. The results obtained showed that while each virus displayed a unique infection phenotype, EIV/2003 exhibited the highest overall fitness, consistent with the long-term circulation of this lineage among horses. Traits associated with increased fitness included enhanced viral replication, efficient cell-to-cell spread in cells and tissues, and resistance to interferon. Notably, transcriptomics revealed important differences among EIVs in terms of intracellular pathways affecting host immunity, inflammation, and cellular transcription. This study showed that within-host fitness is determined by the interplay between virus-host interactions and evolution. In turn, within-host fitness will likely impact long-term adaptation of IAV to mammals
