Phylogenetically estimated neutral rates and fitness effects of mutations to influenza proteins

Influenza virus’s rapid evolution is shaped by both neutral mutation and selection. Phylogenetics can be used to study these processes, but this approach has typically only been applied to a few thousand influenza genome sequences at once. Here, we built phylogenetic trees with >100,000 influenza sequences, and then used these trees to estimate neutral rates of mutations to the virus’s genome. Neutral rates varied by up to ~100-fold among the 12 nucleotide mutation types ( A → C , A → G , etc.). These rates were highly correlated among influenza, SARS-CoV-2, and HIV, though more nuanced context-dependent patterns showed marked differences between influenza and SARS-CoV-2. We also estimated fitness effects of mutations by comparing the number of times a mutation was observed to occur along the branches of a tree to the number of times we expect it to have occurred under neutrality. We estimated effects for ~33,000 nonsynonymous and ~8,000 synonymous mutations spanning all influenza proteins. This compendium of estimated effects helps map the relationship between sequence and fitness in a natural setting, including regions where synonymous mutations are under functional constraint, and for proteins with limited experimentally measured effects. We built interactive heatmaps of the estimated fitness effects to help readers explore these data (see https://matsen.group/flu-mut-rates ). Altogether, this work places influenza’s mutation rates in a broader cross-viral context and deepens our understanding of how mutation and selection shape influenza evolution in nature at a site-specific level.