causing human infection: phylogenetic, structural, and coalescent analyses

Methods We downloaded H7N9 virus genome sequences from the Global Initiative on Sharing Avian Infl uenza Data (GISAID) database and public sequences used from the Infl uenza Virus R esource. We constructed phylogenetic trees and did 1000 bootstrap replicates for each tree. Two rounds of phylogenetic analyses were done. We used at least 100 closely related sequences for each gene to infer the overall topology, removed suspicious sequences from the trees, and focused on the closest clades to the novel H7N9 viruses. We compared our tree topologies with those from a bayesian evolutionary analysis by sampling trees (BEAST) analysis. We used the bayesian Markov chain Monte Carlo method to jointly estimate phylogenies, divergence times, and other evolutionary parameters for all eight gene fragments. We used sequence alignment and homology-modelling methods to study specifi c mutations regarding phenotypes, specifi cally addressing the human receptor binding properties. Findings The novel avian infl uenza A H7N9 virus originated from multiple reassortment events. The HA gene might have originated from avian infl uenza viruses of duck origin, and the NA gene might have transferred from migratory birds infected with avian infl uenza viruses along the east Asian fl yway. The six internal genes of this virus probably originated from two diff erent groups of H9N2 avian infl uenza viruses, which were isolated from chickens. Detailed analyses also showed that ducks and chickens probably acted as the intermediate hosts leading to the emergence of this virulent H7N9 virus. Genotypic and potential phenotypic diff erences imply that the isolates causing this outbreak form two separate subclades.