Influenza human challenge reveals heterogeneous expulsion of infectious virus into the air

Efficient human-to-human spread of influenza viruses requires their release into the air. Owing to long-standing challenges with detecting infectious virus in the air, this process is poorly understood. Here, we describe a methodology to quantify and genotype infectious virus expelled by humans experimentally infected with a seasonal influenza virus. Central to our approach is the Modular Influenza Sampling Tunnel (MIST), designed to capture expulsions from the nose and mouth. Cell monolayers along the MIST's 97 cm length enable culture, quantification and sequencing of infectious virus, while water-sensitive paper and fine particle samplers yield respiratory particle counts over a broad size range. Using MIST, infectious virus was captured from seven of eight infected participants between 2 and 6 days post-inoculation. Airborne virus varied in titer over three orders of magnitude and comprised many genetic variants. Importantly, expulsion of infectious virus did not correlate with viral titers in nasopharyngeal swabs; rather, associations were observed with saliva viral load and clinical symptoms. These data identify the oral cavity as a potential source of transmitted virus, reveal the maintenance of viral diversity through expulsion, and highlight the potential for heterogeneity in expulsion to drive variation in secondary attack rates.