Influenza Virus Infectivity Is Retained in Aerosols and Droplets Independent of Relative Humidity

Abstract

Pandemic and seasonal influenza viruses can be transmitted through aerosols and droplets, in which viruses must remain stable and infectious across a wide range of environmental conditions. Using humidity-controlled chambers, we studied the impact of relative humidity on the stability of 2009 pandemic influenza A(H1N1) virus in suspended aerosols and stationary droplets. Contrary to the prevailing paradigm that humidity modulates the stability of respiratory viruses in aerosols, we found that viruses supplemented with material from the apical surface of differentiated primary human airway epithelial cells remained equally infectious for 1 hour at all relative humidities tested. This sustained infectivity was observed in both fine aerosols and stationary droplets. Our data suggest, for the first time, that influenza viruses remain highly stable and infectious in aerosols across a wide range of relative humidities. These results have significant implications for understanding the mechanisms of transmission of influenza and its seasonality.

Figure 1.

Design of a controlled relative humidity (RH) rotating drum for the aerosolization of influenza viruses. Schematic (top) and photograph (bottom) of the tunable RH rotating drum. The rotating drum (1) is preconditioned to the desired RH prior to aerosolization of the virus. Bulk virus solution is kept on ice and aerosolized via a nebulizer (2) into the drum. The drum is sealed during incubation of the viral aerosols at each specified RH. Viral aerosols are extracted through the sampling port (3) onto a gelatin filter, using a pump, at 2 L/minute for 15 minutes. The gelatin filter is dissolved in warm medium to allow for titration of infectious virus. HEPA, high-efficiency particulate air filter.

Figure 2.

Influenza virus maintains infectivity in fine aerosols at all relative humidities (RHs). A, Schematic representing preparation of the virus in physiological aerosolization medium including extracellular material (ECM) produced by primary human bronchial epithelial (HBE) cells. Inset is a hematoxylin and eosin–stained image of HBE cells, demonstrating the 3-dimensional culture. B, Infectivity of aerosolized 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) supplemented with HBE ECM from uninfected cells in L-15 tissue culture medium. The amount of virus before and after 1 hour of aging in aerosols was determined by a 50% tissue culture infective dose (TCID₅₀) assay on Madin-Darby canine kidney cells. Data represent mean values ± standard deviations of 3 independent biological replicates, exclusive of 85% RH, which was done twice.

Figure 3.

Presence of primary human bronchial epithelial (HBE) cell extracellular material (ECM) protects φ6 bacteriophage from decay at mid-range relative humidities (RHs). A, Infectivity of aerosolized φ6 in tryptic soy broth (TSB) was tested at a range of RHs within the rotating drum. B, As with 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09), φ6 was aerosolized in medium containing HBE ECM for comparison of virus titer in unaged aerosols and aerosols aged for 1 hour. The amount of virus before and after aging was determined by plaque assay. Data represent mean values ± standard deviations of 3 independent biological replicates. PFU, plaque-forming units.

Figure 4.

Viral decay corrected for physical loss of aerosols within the rotating drum. A, Log₁₀ decay of 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) aerosolized with primary human bronchial epithelial (HBE) cell extracellular material (ECM) was calculated as the difference in log₁₀ titer between aged and unaged samples at each relative humidity (RH). A mass balance equation was used to correct for physical loss of aerosols due to gravitational settling and dilution. B, Log₁₀ decay of φ6 in traditional laboratory medium (black) and HBE ECM (blue) demonstrates protection from decay at 75% and 85% RH. PFU, plaque-forming units; TCID₅₀, 50% tissue culture infective dose; TSB, tryptic soy broth.

Figure 5.

Exogenous primary human bronchial epithelial (HBE) cell extracellular material (ECM) protects influenza virus from decay in stationary droplets. The viability of 2009 pandemic influenza A(H1N1) virus (A[H1N1]pdm09) was tested in stationary droplets at a range of different relative humidities (RHs) in a controlled RH chamber. Virus samples were compared with (circles) and without (squares) exogenous HBE ECM. A, RH-dependent decay of A(H1N1)pdm09 with (red) and without (black) exogenous HBE ECM after 1 hour. Data represent mean values ± standard deviations from 3 biological replicates. B, Protection from decay of A(H1N1)pdm09 in droplets is dependent on the concentration of HBE ECM. Virus was prepared with (shaded circles) or without (open circles) HBE ECM dilutions in L-15 tissue culture medium. Droplets were incubated at 55% RH for 1 hour. Individual data points are shown, with error bars indicating standard deviations, and are representative of at least 2 biological replicates. TCID₅₀, 50% tissue culture infective dose.