Emergence of genetically and antigenically diverse strains of influenza to which the human population has no or limited immunity necessitates continuous risk assessments to determine the likelihood of these viruses acquiring adaptations that facilitate sustained human-to-human transmission. As the North American swine H1 virus population has diversified over the last century by means of both antigenic drift and shift, in vivo assessments to study multifactorial traits like mammalian pathogenicity and transmissibility of these emerging influenza viruses are critical. In this review, we examine genetic, molecular, and pathogenicity and transmissibility data from a panel of contemporary North American H1 subtype swine-origin viruses isolated from humans, as compared to H1N1 seasonal and pandemic viruses, including the reconstructed 1918 virus. We present side-by-side analyses of experiments performed in the mouse and ferret models using consistent experimental protocols to facilitate enhanced interpretation of in vivo data. Contextualizing these analyses in a broader context permits a greater appreciation of the role that in vivo risk assessment experiments play in pandemic preparedness. Collectively, we find that despite strain-specific heterogeneity among swine-origin H1 viruses, contemporary swine viruses isolated from humans possess many attributes shared by prior pandemic strains, warranting heightened surveillance and evaluation of these zoonotic viruses.

The ferret transmission model is routinely used to evaluate the pandemic potential of newly emerging influenza A viruses. However, concurrent measurement of viral load in the air is typically not a component of such studies. To address this knowledge gap, we measured the levels of virus in ferret nasal washes as well as viral RNA emitted into the air for 14 diverse influenza viruses, encompassing human-, swine-, and avian-origin strains. Here we show that transmissible viruses display robust replication and fast release into the air. In contrast, poorly- and non-transmissible viruses show significantly reduced or delayed replication along with lower detection of airborne viral RNA at early time points post inoculation. These findings indicate that efficient ferret-to-ferret transmission via the air is directly associated with fast emission of virus-laden particles; as such, quantification of viral RNA in the air represents a useful addition to established assessments of new influenza virus strains.

Each time a swine influenza virus transmits to a human, it provides an opportunity for the virus to acquire adaptations needed for sustained human-to-human transmission. Here, we use aerobiology techniques to test the stability of swine-origin H1 subtype viruses in aerosols and evaluate their infectivity in ferrets. Our results show that highly transmissible influenza viruses display enhanced stability in an aerosol state compared to viruses that do not transmit as efficiently. Similar to human-adapted strains, swine-origin influenza viruses are infectious in ferrets at low doses even after prolonged suspension in the air. These data underscore the risk of airborne swine-origin influenza viruses and support the need for continued surveillance and refinement of innovative laboratory methods to investigate mammalian exposure to inhaled pathogens. Determination of the molecular markers that affect the longevity of airborne influenza viruses will improve our ability to quickly identify emerging strains that present the greatest threat to public health.

Recent A(H5N1) zoonotic cases linked to poultry and cattle in North America highlight the urgent need to assess the pandemic potential of emerging strains. Using male ferrets, we evaluate two B3.13 and two D1.1 genotype A(H5N1) viruses isolated from humans and observe fatal disease and varying capacities for direct contact transmission. To enhance pandemic risk assessment, we conduct aerosol sampling using cyclone BC251 and water condensation capture-based SPOT samplers and perform comparative analyses to include additional A(H5N1), A(H9N2), A(H7N9), and A(H1N1)pdm09 strains with known transmissibility profiles. Although none of the A(H5N1) strains transmit via the air, B3.13 viruses are detected at significantly higher levels compared to D1.1 strains. Here we show strong correlations between viral loads in nasal washes, airborne virus shedding, and transmissibility in ferrets, highlighting the value of these metrics for identifying zoonotic influenza viruses that may be adapting toward increased transmission potential.

Airborne transmission of viral pathogens is dependent on the generation, exhalation, and deposition of virus-laden aerosols from infected to susceptible hosts. Rigorous evaluations of virus transmissibility in laboratory settings have provided critical insight into viral and host features that contribute to this property, but historically have not included concurrent evaluations of viral load in the air. Similarly, investigation of respiratory pathogens in an aerosol state has been understudied relative to other areas focused on transmission dynamics between mammalian hosts. What makes collection and quantification of virus-laden aerosols in the laboratory so challenging? Here, we discuss practical obstacles and limitations on performing this work in laboratory environments, the additional challenges posed by conducting these experiments concurrent with in vivo experimentation, and how continued investment in this work will provide greater understanding of the role aerosols play in viral transmission.