Dynamic Convergent Evolution Drives the Passage Adaptation across 48 Years' History of H3N2 Influenza Evolution.

Influenza viruses are often propagated in a diverse set of culturing media and additional substitutions known as passage adaptation can cause extra evolution in the target strain, leading to ineffective vaccines. Using 25,482 H3N2 HA1 sequences curated from Global Initiative on Sharing All Influenza Data and National Center for Biotechnology Information databases, we found that passage adaptation is a very dynamic process that changes over time and evolves in a seesaw like pattern. After crossing the species boundary from bird to human in 1968, the influenza H3N2 virus evolves to be better adapted to the human environment and passaging them in embryonated eggs (i.e., an avian environment) leads to increasingly stronger positive selection. On the contrary, passage adaptation to the mammalian cell lines changes from positive selection to negative selection. Using two statistical tests, we identified 19 codon positions around the receptor binding domain strongly contributing to passage adaptation in the embryonated egg. These sites show strong convergent evolution and overlap extensively with positively selected sites identified in humans, suggesting that passage adaptation can confound many of the earlier studies on influenza evolution. Interestingly, passage adaptation in recent years seems to target a few codon positions in antigenic surface epitopes, which makes it difficult to produce antigenically unaltered vaccines using embryonic eggs. Our study outlines another interesting scenario whereby both convergent and adaptive evolution are working in synchrony driving viral adaptation. Future studies from sequence analysis to vaccine production need to take careful consideration of passage adaptation.