Comparison of five bacteriophages as models for viral aerosol studies

Abstract

Bacteriophages are perceived to be good models for the study of airborne viruses because they are safe to use, some of them display structural features similar to those of human and animal viruses, and they are relatively easy to produce in large quantities. Yet, only a few studies have investigated them as models. It has previously been demonstrated that aerosolization, environmental conditions, and sampling conditions affect viral infectivity, but viral infectivity is virus dependent. Thus, several virus models are likely needed to study their general behavior in aerosols. The aim of this study was to compare the effects of aerosolization and sampling on the infectivity of five tail-less bacteriophages and two pathogenic viruses: MS2 (a single-stranded RNA [ssRNA] phage of the Leviviridae family), Φ6 (a segmented double-stranded RNA [dsRNA] phage of the Cystoviridae family), ΦX174 (a single-stranded DNA [ssDNA] phage of the Microviridae family), PM2 (a double-stranded DNA [dsDNA] phage of the Corticoviridae family), PR772 (a dsDNA phage of the Tectiviridae family), human influenza A virus H1N1 (an ssRNA virus of the Orthomyxoviridae family), and the poultry virus Newcastle disease virus (NDV; an ssRNA virus of the Paramyxoviridae family). Three nebulizers and two nebulization salt buffers (with or without organic fluid) were tested, as were two aerosol sampling devices, a liquid cyclone (SKC BioSampler) and a dry cyclone (National Institute for Occupational Safety and Health two-stage cyclone bioaerosol sampler). The presence of viruses in collected air samples was detected by culture and quantitative PCR (qPCR). Our results showed that these selected five phages behave differently when aerosolized and sampled. RNA phage MS2 and ssDNA phage ΦX174 were the most resistant to aerosolization and sampling. The presence of organic fluid in the nebulization buffer protected phages PR772 and Φ6 throughout the aerosolization and sampling with dry cyclones. In this experimental setup, the behavior of the influenza virus resembled that of phages PR772 and Φ6, while the behavior of NDV was closer to that of phages MS2 and ΦX174. These results provide critical information for the selection of appropriate phage models to mimic the behavior of specific human and animal viruses in aerosols.

FIG 1

Effect of nebulizer and nebulization buffer on relative recoveries of phage PR772 by qPCR and culture. Samples were taken with the SKC BioSampler (black symbols) or the NIOSH two-stage cyclone bioaerosol sampler at 10 liters/min (gray symbols). The black and gray bars indicate the median relative recoveries obtained with each sampler (n = 3). §, P = 0.03 for relative recovery by qPCR and P < 0.001 for relative recovery by culture (PFU) for comparison of the SKC BioSampler and the NIOSH two-stage cyclone bioaerosol sampler; *, P < 0.05 for comparison of the relative recoveries obtained with both air samplers when samples were aerosolized with different nebulizers.

FIG 2

Comparison of relative recovery by qPCR and relative recovery by culture of the five phage models nebulized all together and sampled with the SKC BioSampler (black symbols) and the NIOSH two-stage cyclone bioaerosol sampler at 3.5 liters/min (dark gray symbols) and 10 liters/min (light gray symbols). The bars indicate the median relative recoveries obtained with each sampler (n = 6). P values were determined by an unpaired t test of normally distributed log-transformed data. *, comparison of the SKC BioSampler and the NIOSH two-stage cyclone bioaerosol sampler at 3.5 liters/min and 10 liters/min for relative recovery by culture. P was <0.0001 for phage ΦX174. The use of a in the left panel indicates that the relative recoveries by qPCR for these phages were not significantly different. The use of a also indicates that the qPCR and culture relative recoveries were not significantly different for phage MS2 with all air samplers. For a comparison of the qPCR and culture relative recoveries with all air samplers, P was <0.0001 for phages PM2, Φ6, PR772, and ΦX174. a, b, c, and d, comparison of the relative recoveries of phages by culture (PFU). P was <0.05 with the NIOSH two-stage cyclone bioaerosol sampler at 10 liters/min when comparing phages in cluster b with phages in clusters a, c, and d. P was <0.05 between clusters c and a with all air samplers. P was <0.05 between clusters c and d with the SKC BioSampler. P was <0.05 between clusters a and d with all air samplers.

FIG 3

Comparison of the relative recovery by culture of phage Φ6 aerosolized from phage buffer or phage buffer supplemented with organic fluid and sampled using the NIOSH two-stage cyclone bioaerosol sampler at 3.5 liters/min (dark gray symbols) and 10 liters/min (light gray). The bars indicate the median relative recoveries obtained with each sampler (n = 3). *, P = 0.0018 by comparison of the two nebulization buffers with air sampling using the NIOSH two-stage cyclone bioaerosol sampler. An unpaired t test was performed on normally distributed log-transformed data.

FIG 4

Comparison of the relative culture/genome ratios for the five phage models with those for influenza A virus H1N1 (InfA) and NDV. The viruses were sampled with the SKC BioSampler. The bars indicate the median relative culture/genome ratio obtained for each virus. The relative ratios of PFU obtained by culture to genomes obtained by PCR could not be calculated for phage PM2 because no plaques could be detected for this phage under the conditions used in the six experiments. Data are for six experiments for all phages and three experiments for influenza A virus and NDV. a and b, comparison of the relative culture/genome ratios of the viruses. An unpaired t test was performed on normally distributed log-transformed data. P was >0.05 between viruses in cluster a, and P was >0.05 between the viruses in cluster b. P was <0.05 between the viruses in clusters a and b.