Development of an improved methodology to detect infectious airborne influenza virus using the NIOSH bioaerosol sampler

Abstract

A unique two-stage cyclone bioaerosol sampler has been developed at NIOSH that can separate aerosols into three size fractions. The ability of this sampler to collect infectious airborne viruses from a calm-air chamber loaded with influenza A virus was tested. The sampler's efficiency at collecting aerosolized viral particles from a calm-air chamber is essentially the same as that from the high performance SKC BioSampler that collects un-fractionated particles directly into a liquid media (2.4 × 10(4) total viral particles per liter of sampled air (TVP/L) versus 2.6 × 10(4) TVP/L, respectively, after 15 min) and the efficiency is relatively constant over collection times of 15, 30 and 60 min. Approximately 34% of the aerosolized infectious virus collected after 15 min with the NIOSH bioaerosol sampler remained infectious, and infectious virus was found in all three size fractions. After 60 min of sampling, the infectious virus/liter air found in the NIOSH bioaerosol sampler was 15% of that found in the SKC BioSampler. This preservation of infectivity by the NIOSH bioaerosol sampler was maintained even when the initial infectivity prior to aerosolization was as low as 0.06%. The utility of the NIOSH bioaerosol sampler was further extended by incorporating an enhanced infectivity detection methodology developed in our laboratory, the viral replication assay, which amplified the infectious virus making it more readily detectable.

Fig. 1. Description of the NIOSH Bioaerosol Sampler

Ambient air is drawn into the sampler’s inlet at 3.5 L min⁻¹ and initially enters the 1st stage 15 ml polypropylene tube, where aerosol particles >4 µm are deposited on the wall of the tube. The air then enters the 2nd stage 1.5 ml polypropylene tube where 1 to 4 µm particles are deposited. After the air exits the 2nd stage, particles <1 µm are collected on a 37 mm PTFE filter.

Fig. 2. Description of the Calm-air Chamber

Influenza virus is loaded into a nebulizer, mixed with dry air, and the aerosolized particles are dispersed into an air chamber. NIOSH bioaerosol samplers are placed into the bottom of the chamber and an SKC BioSampler is placed outside the chamber.

Fig. 3. Infectious influenza virus detected in the NIOSH bioaerosol sampler

The amount of infectious influenza virus detected in each stage per liter of air collected is shown here relative to the amount detected in the SKC BioSampler. After 15, 30 and 60 min, the infectious virus/liter air found in the NIOSH bioaerosol sampler was 34%, 28% and 15% of that found in the SKC BioSampler.

Fig. 4. Size distribution of influenza-laden aerosol particles in the calm-air chamber

The plot shows the estimated mass of aerosol particles vs. particle aerodynamic diameter for a typical experiment as measured by an Aerodynamic Particle Sizer. If the viral particles are evenly distributed in the nebulized droplets, the mass is proportional to the viral content. Over the range of the APS, the test aerosol containing HBSS and influenza virus had an average concentration of 1.33 × 10⁶ particles/cm², with a count median aerodynamic diameter of 0.8 µm and a geometric standard deviation of 1.26. The HBSS-only aerosol had an average concentration of 1.20 × 10⁶ particles/cm², with a count median diameter of 0.7 µm and a geometric standard deviation of 1.23.