Mimicking evolution to define mechanisms of airborne transmission of H7N9 viruses

The Asian lineage H7N9 avian influenza viruses (AIV) have caused >1500 human zoonotic infections with 615 deaths. These viruses have not spread in humans; however, there is a high potential for these viruses to evolve to transmit via the airborne route and cause a pandemic. Using ferrets, we previously evaluated the ability of the prototypic Asian lineage virus, A/Anhui/1/2013 (H7N9), to undergo two continuous rounds of airborne transmission. In these studies, we found that the virus was able to transmit to 50-66% of respiratory contact ferrets during both rounds of transmission. In a subsequent deep sequence analysis, we identified 2-5 mutations in 90-99% of all variant viruses that transmitted. These mutations were in the hemagglutinin (HA), neuraminidase (NA), and viral polymerase genes. As airborne transmission is associated with enhanced binding and replication in cells of the upper airways, we hypothesize that the identified mutations will alter the molecular properties of the virus to enhance replication in primary human nasal and tracheal epithelial cells. Our aims are: Aim 1. Determine the role of previously identified HA and NA mutations in an H7N9 virus with the A/PR/8 vaccine backbone. Viruses carrying the H7N9 HA and NA on the A/PR8 vaccine backbone will generated. Mutations will be introduced into the HA and NA gene segments and several properties including receptor-binding preference, pH of fusion, thermostability, NA activity, and changes in antibody recognition via immune serum will be evaluated. Aim 2. Evaluate the role of previously identified mutations on the viral polymerase. To assess the impact of mutations in the viral polymerase, in vitro polymerase reconstitution assays will be performed. Specifically, the activity of the wild-type H7N9 polymerase with and without the identified mutations will be assessed. Aim 3. Determine if the introduction of previously identified mutations alters viral replication in primary human airway epithelial cells. To determine if the identified mutations impact viral replication, we will evaluate the replication kinetics of recombinant H7N9-A/PR8 viruses for their growth in primary human airway epithelial cells. Primary human cells will include nasal, tracheal, bronchial, and small airway epithelial cells. Collectively, these studies will determine the effect of the identified mutations on different molecular properties of the virus, while also determining if the mutations alter the viral tropism in human cells. Our findings will generate new insight on how AIV evolve to transmit via the airborne route and will yield critical knowledge required to interpret the evolution and assess the pandemic potential of H7N9 viruses.