Daniel Verreault

Methods for Sampling of Airborne Viruses

SUMMARY To better understand the underlying mechanisms of aerovirology, accurate sampling of airborne viruses is fundamental. The sampling instruments commonly used in aerobiology have also been used to recover viruses suspended in the air. We reviewed over 100 papers to evaluate the methods currently used for viral aerosol sampling. Differentiating infections caused by direct contact from those caused by airborne dissemination can be a very demanding task given the wide variety of sources of viral aerosols. While epidemiological data can help to determine the source of the contamination, direct data obtained from air samples can provide very useful information for risk assessment purposes. Many types of samplers have been used over the years, including liquid impingers, solid impactors, filters, electrostatic precipitators, and many others. The efficiencies of these samplers depend on a variety of environmental and methodological factors that can affect the integrity of the virus structure. The aerodynamic size distribution of the aerosol also has a direct effect on sampler efficiency. Viral aerosols can be studied under controlled laboratory conditions, using biological or nonbiological tracers and surrogate viruses, which are also discussed in this review. Lastly, general recommendations are made regarding future studies on the sampling of airborne viruses.

Detection of Airborne Lactococcal Bacteriophages in Cheese Manufacturing Plants

ABSTRACT The dairy industry adds starter bacterial cultures to heat-treated milk to control the fermentation process during the manufacture of many cheeses. These highly concentrated bacterial populations are susceptible to virulent phages that are ubiquitous in cheese factories. In this study, the dissemination of these phages by the airborne route and their presence on working surfaces were investigated in a cheese factory. Several surfaces were swabbed, and five air samplers (polytetrafluoroethylene filter, polycarbonate filter, BioSampler, Coriolis cyclone sampler, and NIOSH two-stage cyclone bioaerosol personal sampler) were tested. Samples were then analyzed for the presence of two Lactococcus lactis phage groups (936 and c2), and quantification was done by quantitative PCR (qPCR). Both lactococcal phage groups were found on most swabbed surfaces, while airborne phages were detected at concentrations of at least 103 genomes/m3 of air. The NIOSH sampler had the highest rate of air samples with detectable levels of lactococcal phages. This study demonstrates that virulent phages can circulate through the air and that they are ubiquitous in cheese manufacturing facilities.

Airborne porcine circovirus in Canadian swine confinement buildings

Porcine circovirus type 2 has been linked to many diseases, such as postweaning multisystemic wasting syndrome and can be found in most commercial swine confinement buildings around the world. Although the exact role of the virus in the appearance of disease in animals is not fully understood, the mechanisms responsible for the transmission of the virus are currently believed to happen mostly by contact. Nevertheless, the possibility of airborne transmission cannot be rejected. This study investigated the presence of the virus, total bacteria and total dusts in aerosols. Air samples were taken with gelatin filters in swine confinement buildings and were analyzed by quantitative polymerase chain reaction. Interestingly, concentrations of airborne PCV2 of up to 10(7) genomes per cubic meter of air were detected. Airborne dust concentrations were correlated to airborne concentrations of PCV2 and total bacteria. Although the infectivity potential of the airborne viral loads were not evaluated, it is clear that the virus can become airborne in detectable concentrations in commercial swine confinement building environments. The significance of this finding in an epidemiological point of view will need further investigation.

Comparison of Polycarbonate and Polytetrafluoroethylene Filters for Sampling of Airborne Bacteriophages

Aerosolized coliphage phiX174 and lactococcal phage P008 were sampled with two types of filter, polycarbonate (PC) and polytetrafluoroethylene (PTFE). The recovery was determined by plaque assays and quantitative polymerase chain reaction (qPCR). Recovery by qPCR was greater than by culture and PC filters outperformed PTFE filters both by culture and by qPCR relative recovery. The results of the plaque assays showed that the infectivity of the recovered phages was affected by the aerosolization/air sampling. The presence of viruses in air samples should be determined by culture-independent assays.

Evaluation of Filters for the Sampling and Quantification of RNA Phage Aerosols

This study exploits the virulent bacteriophages phi 6 (dsRNA) and MS2 (ssRNA) as surrogates for airborne RNA viruses. Two different filter types, polytetrafluoroethylene (PTFE) and polycarbonate (PC), were tested for their efficiency in collecting aerosolized RNA phages. Two commercial kits were tested for total RNA isolation. Also, heat shock treatments were performed in three different media to obtain the most favorable conditions for reverse transcription assays of dsRNA. Our findings suggest that PC filters are more suitable to recover infectious airborne RNA viruses as determined by plaque assays. Both types of filters were equally efficient in recovering RNA from aerosolized phage phi 6 as established by qRT-PCR. Viral samples should be treated with QIAamp Viral RNA Mini Kit and a 5 min heat shock treatment at 110°C in TE buffer before RT-PCR to maximize detection of phage phi 6. Overall, the infectivity of the recovered phages was severely affected by the aerosolization/air sampling process and the presence of RNA viruses in air samples should be determined by qRT-PCR.

Resistance of Aerosolized Bacterial Viruses to Relative Humidity and Temperature.

ABSTRACT The use of aerosolized bacteriophages as surrogates for hazardous viruses might simplify and accelerate the discovery of links between viral components and their persistence in the airborne state under diverse environmental conditions. In this study, four structurally distinct lytic phages, MS2 (single-stranded RNA [ssRNA]), ϕ6 (double-stranded RNA [dsRNA]), ϕX174 (single-stranded DNA [ssDNA]), and PR772 (double-stranded DNA [dsDNA]), were nebulized into a rotating chamber and exposed to various levels of relative humidity (RH) and temperature as well as to germicidal UV radiation. The aerosolized viral particles were allowed to remain airborne for up to 14 h before being sampled for analysis by plaque assays and quantitative PCRs. Phages ϕ6 and MS2 were the most resistant at low levels of relative humidity, while ϕX174 was more resistant at 80% RH. Phage ϕ6 lost its infectivity immediately after exposure to 30°C and 80% RH. The infectivity of all tested phages rapidly declined as a function of the exposure time to UVC radiation, phage MS2 being the most resistant. Taken altogether, our data indicate that these aerosolized phages behave differently under various environmental conditions and highlight the necessity of carefully selecting viral simulants in bioaerosol studies.

Flow Cytometry Sorting Protocol of Bacillus Spore Using Ultraviolet Laser and Autofluorescence as Main Sorting Criterion

The ultraviolet (UV) Fluorescent Aerodynamic Particle Sizer (FLAPS), a flow cytometer-like apparatus was developed by the Canadian Department of National Defence for real-time detection of autofluorescence of biological aerosol particles such as bacterial spores. The direct relation between autofluorescence intensity and viability has recently been reported and viable spore are more autofluorescent in UV (Laflamme, Frontiers in Bioscience). The goal of this manuscript is to describe a flow cytometry sorting protocol based on UV-induced autofluorescence. An EPICS® ELITE ESP flow cytometer equipped with a UV laser and cell sorter was used to mimic the optical properties of FLAPS and to study the two extremes of a spore population according to its autofluorescence (lower level of autofluorescence (LLA) and higher level of autofluorescence (HLA) spores). Bacillus subtilis var niger was used as a surrogate for Bacillus anthracis spores and sorted using autofluorescence intensity as the main criterion. The protocol developed in our laboratory to sort Bacillus spores according to their autofluorescence properties is described. Purity of each sorted population was greater than 95%. Using autofluorescence as the main criterion, we demonstrate that it is possible to separate two distinct spore populations.

Design of an environmentally controlled rotating chamber for bioaerosol aging studies

Abstract A chamber was designed and built to study the long-term effects of environmental conditions on air-borne microorganisms. The system consists of a 55.5-L cylindrical chamber, which can rotate at variable speeds on its axis. The chamber is placed within an insulated temperature controlled enclosure which can be either cooled or heated with piezoelectric units. A germicidal light located at the chamber center irradiates at a 360° angle. Access ports are located on the stationary sections on both ends of the chamber. Relative humidity (RH) is controlled by passing the aerosol through meshed tubes surrounded by desiccant. Validation assay indicates that the interior temperature is stable with less than 0.5 °C in variation when set between 18 and 30 °C with the UV light having no effect of temperature during operation. RH levels set at 20%, 50% and 80% varied by 2.2%, 3.3% and 3.3%, respectively, over a 14-h period. The remaining fraction of particles after 18 h of suspension was 8.8% at 1 rotation per minute (rpm) and 2.6% at 0 rpm with the mass median aerodynamic diameter (MMAD) changing from 1.21 ± 0.04 µm to 1.30 ± 0.02 µm at 1 rpm and from 1.21 ± 0.04 µm to 0.91 ± 0.01 µm at 0 rpm within the same time period. This chamber can be used to increase the time of particle suspension in an aerosol cloud and control the temperature, RH and UV exposure; the design facilitates stationary sampling to be performed while the chamber is rotating.