A naturally occurring HA-stabilizing amino acid (HA1-Y17) in an A(H9N2) low-pathogenic influenza virus contributes to airborne transmission

ABSTRACT Airborne transmissibility is a prerequisite for a pandemic influenza A virus (IAV), and a better understanding of how zoonotic IAV evolves to acquire a transmissible phenotype is essential for pandemic preparedness. Select contemporary influenza A(H9N2) viruses such as A/Anhui-Lujiang/39/2018 (AL/39) have exhibited a limited transmission capability by the airborne route in the ferret model; therefore, it is of great importance to identify viral factors that contribute to enhanced transmission. To investigate the role of virus acid stability in virus airborne transmission, we rescued a pair of isogenic A(H9N2) viruses, including the wild-type (wt) AL/39 and the mutant virus bearing a naturally occurring substitution HA1-Y17H, with a resulting difference in virus pH thresholds for hemagglutinin activation. We next assessed virus replication, airborne transmission, and fitness in a co-infection competition model in ferrets. We found that the HA1-Y17H mutant virus yielded only non-productive airborne transmission despite possessing a comparative replication as the wt virus in the ferret upper respiratory tract. Furthermore, ferrets inoculated with the wt virus emitted more virus-laden particles into the air than the HA1-Y17H mutant virus-inoculated animals. During ferret co-infection experiments, the wt virus was the dominant species in multiple types of specimens following different inoculation routes. Taken together, our study demonstrates that an acid-stable IAV had a greater capacity to establish a productive infection in the ferret upper respiratory tract and was emitted in greater quantities from infected animals, features that may contribute to virus airborne transmission in a synergistic manner in mammalian hosts. IMPORTANCE Despite the accumulation of evidence showing that airborne transmissible influenza A virus (IAV) typically has a lower pH threshold for hemagglutinin (HA) fusion activation, the underlying mechanism for such a link remains unclear. In our study, by using a pair of isogenic recombinant A(H9N2) viruses with a phenotypical difference in virus airborne transmission in a ferret model due to an acid-destabilizing mutation (HA1-Y17H) in the HA, we demonstrate that an acid-stable A(H9N2) virus possesses a multitude of advantages over its less stable counterpart, including better fitness in the ferret respiratory tract, more effective aerosol emission from infected animals, and improved host susceptibility. Our study provides supporting evidence for the requirement of acid stability in efficient airborne transmission of IAV and sheds light on fundamental mechanisms for virus airborne transmission.