Three waves of human infection with H7N9 influenza viruses have concluded to date, but only viruses within the first wave (isolated between March-September 2013) have been extensively studied in mammalian models. While second- and third-wave viruses remain closely linked phylogenetically and antigenically, even subtle molecular changes can impart critical shifts in mammalian virulence. To determine if H7N9 viruses isolated from humans during 2013-15 have maintained the phenotype first identified among 2013 isolates, we assessed the ability of first-, second-, and third-wave H7N9 viruses isolated from humans to cause disease in mice and ferrets and to transmit among ferrets. Similar to first-wave viruses, H7N9 viruses from 2013-15 were highly infectious in mice, with comparable lethality to the well-studied A/Anhui/1/2013 virus. Second- and third-wave viruses caused moderate disease in ferrets, transmitted efficiently to cohoused, na?ve contact animals, and demonstrated limited transmissibility by respiratory droplets. All H7N9 viruses replicated efficiently in human bronchial epithelial cells, with subtle changes in pH fusion threshold identified between H7N9 viruses examined. Our results indicate that despite increased genetic diversity and geographical distribution since their initial detection in 2013, H7N9 viruses have maintained a pathogenic phenotype in mammals and continue to represent an immediate threat to public health.

IMPORTANCE:

H7N9 influenza viruses, first isolated in 2013, continue to cause human infection and represent an ongoing public health threat. Now entering the fourth wave of human infection, H7N9 viruses continue to exhibit genetic diversity in avian hosts, necessitating continuous efforts to monitor their pandemic potential. However, viruses isolated post-2013 have not been extensively studied, limiting our understanding of potential changes in virus-host adaptation. In order to ensure that current research with first-wave H7N9 viruses still pertains to more recently isolated strains, we compared the relative virulence and transmissibility of H7N9 viruses isolated during the second and third waves, through 2015, in the mouse and ferret models. Our finding that second and third wave viruses generally exhibit comparable disease in mammals as first-wave viruses strengthens our ability to extrapolate research from the 2013 viruses to current public health efforts. These data further contribute to our understanding of molecular determinants of pathogenicity, transmissibility, and tropism.