Ancestral sequence reconstruction pinpoints adaptations that enable avian influenza virus transmission in pigs

Here we report adaptative mutations of five recombinant viruses (designated RG-EA1, EA2, EA3, EA4, and RG-Be02-lungSGxEA2IG) in MDCK cells, embryonated chickens eggs, newborn pig tracheal cells (NPTr), and pigs. The recombinant virus RG-EA1, representing the precursor virus of European avian-like H1N1 (EA) swine influenza viruses, was constructed based on the inferred nodal sequences at the split between avian and EA swine H1N1 lineages. RG-EA2 virus was constructed to represent the early evolutionary stage of EA swine viruses, while RG-EA3 and RG-EA4 viruses were constructed at later stages of EA swine virus evolution. RG-Be02-lungSGxEA2IG is a recombinant RG-EA2 virus carrying the HA and NA genes derived from an archived pig lung (A/swine/Belgium/2/1979). We used transfection supernatant as starting materials to passage in parallel viruses in MDCK cells, in eggs, and NPTr cells. MDCK and NPTr cells were infected at the same MOI of 0.001-0.005 and each egg was injected 10e4 pfu in a volume of 0.1 mL. Three independent serial passaging experiments were performed. Supernatant or allantoic fluid harvested from passage three (P3) was analyzed by NGS. Since NP-R351K and PB1-Q621R mutations were under positive selection among EA swine viruses, we focused on these two mutations and compared the transmissibility of RG-EA2NP-R351K and RG-EA2PB 1-Q621R, NP-R351K viruses in pigs. Donor pigs were intranasally inoculated with 10e6 plaque-forming units of virus and were co-housed with naive contact pigs. Next-generation sequencing analyses were performed on the peak-titer nasal swab samples of positive contact pigs.