Aerosolized Influenza Virus: Not Just for Mammalian Models

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In vitro exposure system for study of aerosolized influenza virus

Text by Hannah Creager and Jessica Belser

Humans become infected with respiratory viruses either by inhaling virus or by getting virus on their hands and touching their nose or eyes. People generally do not pour virus-laden liquid (or any liquid, for that matter) down their windpipes. However, traditional methods used for inoculation of cultured cells during in vitro studies are more akin to the latter than the former: cell monolayers are typically covered with virus-containing liquid media and this is allowed to sit for a period of time (often an hour).

Our group had previously used an aerosol exposure system to infect both ferrets and mice with influenza virus, and we wondered whether cultured cells might also be infected in this more natural way. A search of the literature revealed a number of toxicology studies that exposed human respiratory cells to airborne irritants such as cigarette smoke or diesel exhaust, and found that the cells responded differently than when they were incubated with liquid media containing pollutant particles. As no one had done the same with a pathogen, we performed the experiment. To our happy surprise, when we placed cultured cells in our animal exposure chamber and pumped in viral aerosols, they got infected! This raised a critical question: how much virus had the cells been exposed to? The toxicology studies had determined exposure dose based on properties of particles, but we were interested not in the water droplets we aerosolized, but in the viruses within them. After trying several woefully ineffective approaches, we settled on estimating dose using methods more akin to those we use in ferrets—multiplying the dose for the entire exposure chamber by the proportion of the aerosol which came into contact with either the ferret respiratory tract or the cell monolayer. Experimental validation showed this method to be quite effective.

With dosage estimates in hand, we were able to determine that the three different viruses we tested were all able to replicate in human bronchial cells that were exposed to only a handful of virus particles, delivered in either an aerosol or a liquid inoculum. When we used primary human lung cells, however, only one of the viruses was able to replicate productively after aerosol inoculation, even though all three could do so when traditional (ie liquid) inoculation was used. We suspect that something about the aerosol delivery prevents virus from entering cells and look forward to further investigation of virus-cell interactions.


Figure legend

Graphic representation of aerosol system for in vitro use. Figure 1 from manuscript.

Introducing the authors


Hannah Creager is a PhD candidate at Emory University and ORISE fellow at the Centers for Disease Control and Prevention (CDC); Jessica Belser is a microbiologist at the CDC.

About the research

In vitro exposure system for study of aerosolized influenza virus
Hannah M. Creager, Hui Zeng, Joanna A. Pulit-Penaloza, Taronna R. Maines, Terrence M. Tumpey, Jessica A. Belser
Virology, Volume 500, January 2017, Pages 62–70