Long-term adaptation following virus host shifts results in higher replication rate, broader intra-host spread and lower pathogenicity

Influenza A viruses (IAVs) have their main natural reservoir in wild birds and have established in a limited number of mammals, sometimes with devastating consequences. This is illustrated by the four human pandemics that occurred in the last 100 years as well as the large number of outbreaks and epizooties in various species of domestic animals. Infection of a mammal by an avian-origin virus can cause variable outcomes ranging from lethal infections (such as those caused by H5N1 and H7N9 in humans) to outbreaks and pandemics that might lead to the establishment of new endemic viruses. H3N8 equine influenza virus (EIV) constitutes an example of an avian-origin IAV that jumped into a mammalian host -the horse- and established as an endemic lineage that has been circulating in many countries for over 50 years. Virus adaptation requires finely tuned interactions between virus and host proteins, as molecular incompatibilities could act as effective species barriers. We hypothesise that mammalian adaptation is evolutionarily driven and involves multiple changes in virus-host interactions that lead to higher fitness as well as in changes in virus pathogenesis. To test our hypothesis, we infected an interferon-competent equine cell line (E-Derm) with two evolutionarily distinct H3N8 IAVs (A/equine/Uruguay/1/1963 [EIV/63], and A/equine/Ohio/1/2003 [EIV/2003]) and quantified changes in gene expression levels by performing transcriptome analysis at different times post-infection. Cells infected with EIV/63 and EIV/2003 displayed significant differences in gene expression patterns, resulting in virus-specific changes on the regulation of intracellular pathways. Particularly, we observed that cells infected with EIV/2003 exhibit at early time-points a lower number of differentially expressed genes (DEGs) but associated with a high level of regulation. In addition, we observed significant differences between the two EIVs on the virus-induced lesions within the respiratory tract of the horse. In sum, our results provide an initial inventory of genes and intracellular pathways that are likely to be important for the adaptation of avian-origin IAVs to mammals, and also show that IAV evolution has a significant impact on host gene regulation and viral pathogenesis.