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. Author manuscript; available in PMC: 2022 Oct 14.
Published in final edited form as: J Occup Environ Hyg. 2021 Aug 17;18(9):461–475. doi: 10.1080/15459624.2021.1948047

Impact of Sampling and Storage Stress on the Recovery of Airborne SARS-CoV-2 Virus Surrogate Captured by Filtration

Nirmala T Myers 1, Taewon T Han 1, Mei-Ling Li 2, Gary Brewer 2, Martin Harper 3,4, Gediminas Mainelis 1
PMCID: PMC9563239  NIHMSID: NIHMS1828168  PMID: 34197274

Abstract

Environmental air sampling of the SARS-CoV-2 virus in occupational and community settings is pertinent to reduce and monitor the spread of the COVID pandemic. However, there is a general lack of standardized procedures for airborne virus sampling and limited knowledge of how sampling and storage stress impact the recovery of captured airborne viruses. Since filtration is one of the commonly used methods to capture airborne viruses, this study analyzed the effect of sampling and storage stress on SARS-CoV-2 surrogate virus (human coronavirus OC43, or HCoV-OC43) captured by filters.

HCoV-OC43, a simulant of the SARS-CoV-2, was aerosolized and captured by PTFE-laminated filters. The impact of sampling stress was evaluated by comparing the RNA yields recovered when sampled at 3 L/min and 10 L/min and for 10 mins and 60 mins; in one set of experiments, additional stress was added by passing clean air through filters with the virus for 1, 5, and 15 hours. The impact of storage stress was designed to examine RNA recovery from filters at room temperature (25°C) and refrigerated conditions (4°C) for up to one week of storage.

To our knowledge, this is the first report on using HCoV-OC43 aerosol in air sampling experiments, and the mode diameter of the virus aerosolized from the growth medium was 40-60 nm as determined by SMPS + CPC system (TSI Inc.) and MiniWRAS (Grimm Inc.) measurements. No significant difference was found in virus recovery between the two sampling flow rates and different sampling times (p > 0.05). However, storage at room temperature (25°C) yielded ~2x less RNA than immediate processing and storage at refrigerated conditions (4°C). Therefore, it is recommended to store filter samples with viruses at 4°C up to one week if the immediate analysis is not feasible. Although the laminated PTFE filter used in this work purposefully does not include a non-PTFE backing, the general recommendations for handling and storing filter samples with viral particles are likely to apply to other filter types.

Keywords: Airborne Virus Sampling, SARS-CoV-2 Virus Surrogate, Human Coronavirus OC43 (HCoV-OC43), Filtration, Sampling Stress, Storage Stress

2. INTRODUCTION

Occupational and community exposures to the SARS-CoV-2 virus are currently a critical concern worldwide, and the airborne transmission of the virus is a major route of transmission (AIHA 2020; Brosseau et al. 2021; Morawska and Cao 2020; Prather et al. 2020; Samet et al. 2021). Environmental air sampling for the SARS-CoV-2 virus has been performed in multiple environments. In healthcare centers, SARS-CoV-2 RNA was detected in samples taken in airborne infection isolation rooms using NIOSH BC 251 Bioaerosol Samplers (Chia et al. 2020) and the VIVAS Air Sampler (Lednicky et al. 2020); doctors’ office area, and near patients and medical staff areas using the SASS 2300 Wetted Wall Cyclone Sampler (Guo et al. 2020) and a cascade impactor (Liu et al. 2020); central ventilation ducts using exhaust filters (Nissen et al. 2020); and intensive care units, including in corridors of COVID-19 hospital wards with the MD8 Airport Portable Air Sampler containing gelatin membrane filters (Razzini et al. 2020). Additionally, Santarpia et al. (2020) detected viral particles using personal air samplers (Button Aerosol Sampler) with gelatin membrane filters carried by the study personnel in the biocontainment and COVID-19 quarantine units. In non-healthcare environments, SARS-CoV-2 RNA was detected in over 64% of air samples collected in public spaces and transportation in Iran using the AV1000 sampler with PTFE and glass fiber filters (Hadei et al. 2021).

Though researchers worldwide have conducted air sampling for the SARS-CoV-2 virus in multiple investigations, there is a lack of standardized and uniform protocols for airborne virus sampling, storage, and recovery. In general, the virus recovery from a sample depends on the sampling device (filters, impingers, solid impactors, electrostatic precipitators) and collection medium (dry vial, liquid, filter, agar) (NIOSH 2020). Furthermore, depending on the sampler and sampling technology choice, the method of detection and analysis can differ (CDC 2019). Consequently, given the public health importance of airborne viruses, there is a great need to develop and validate protocols used to sample airborne viruses (Verreault et al. 2008). Studies on airborne SARS-CoV-2 utilize a variety of sampling methods, include impactors (Chirizzi et al. 2021), impingers (Faridi et al. 2020), filters (Chia et al., 2020; Liu et al., 2020), and condensational growth devices (Lednicky et al. 2020). Among the different virus sampling techniques, filtration is one of the most commonly used methods as it is easy to use and is compatible with various molecular analysis techniques (Pan et al. 2019b).

Even though filters are widely used to capture airborne viruses, there is limited analysis of how sampling and storage stresses affect the recovery of target RNA. In general, it is assumed that desiccation during sampling and storage can degrade the integrity of nucleic acids captured on filters (Rahmani et al. 2020), thus reducing detection accuracy. On the other hand, long sampling times could be needed to capture a sufficient amount of viral RNA required for reliable analysis. In many instances, the captured samples cannot be processed immediately and have to be stored or shipped to specialized analytical facilities, thus increasing the risk of sample degradation. The need to accurately detect viral RNA captured on filters has become especially acute during the current COVID-19 pandemic when numerous studies aiming to understand the presence and spread of deadly SARS-CoV-2 virus are performed. Thus, the effect of sampling and storage stresses on the ability to elute viral particles captured on filters and determine viral RNA content warrants a detailed investigation.

PTFE is one of the most popular types of filter types used to capture viruses due to high collection efficiency, relatively low-pressure drop, and compatibility with polymerase chain reaction (PCR) (Lindsley et al. 2017). Filters of this type have been used to sample airborne SARS-CoV-2 (Ong et al. 2020; Chia et al. 2020). Recently, Environmental Express, Inc. (Ocala, Florida, USA) has developed a proprietary 37 mm, 1 μm pore-size, PTFE-laminated PTFE ZePore™ filter with a polypropylene support pad in a 3-piece conductive (static-dissipative) plastic cassette (together marketed as the Vira-Pore cassette) that could be used to sample airborne viruses, including SARS-CoV-2. The Vira-Pore cassette can be used for area or personal sampling. Since PTFE filters even with 5 μm pore size have a minimum collection efficiency of over 95% for 300 nm aerodynamic equivalent diameter sodium chloride particles (Soo et al. 2016), one can infer that this filter will have a similar sampling performance. Moreover, filter processing protocols, particularly the elution of the collected airborne virus from this filter, have not been studied previously.

Ideally, examining any technique targeting airborne SARS-CoV-2 should be conducted using this particular virus, but that is not feasible in many cases since it requires proper personal protective equipment (PPE) such as powered air-purifying respirators and specialized facilities, e.g., biosafety level 3 equipped with negative pressure (BSL-3). Therefore, this study used the human coronavirus OC43 (HCoV-OC43; BSL-2), an example of a non-severe coronavirus strain that causes the common cold (Jean et al. 2013), as a simulant of SARS-CoV-2 (BSL-3). Both HCoV-OC43 and SARS-CoV-2 are enveloped betacoronaviruses with positive-sense, single-stranded RNA (+ssRNA) (Vijgen et al. 2005), making HCoV-OC43 a suitable surrogate for studies targeting SARS-CoV-2. Other recent studies have also used HCoV-OC43 as a model to facilitate our understanding of the SARS-CoV-2, though they did not involve aerosolization. Uppal et al. (2021) utilized HCoV-OC43 for testing the inactivation of human coronavirus on PPE and surfaces by a disinfection device. A low-cost method to titrate human coronavirus was developed by Bracci et al. (2020) by utilizing both HCoV-OC43 and HCoV-229E. To our knowledge, the HCoV-OC43 aerosol has not been previously reported in air sampling and validation studies.

The overall goal of this work was to determine the best recovery protocol and the impact of sampling stress (e.g., sampling time and flowrate) and filter storage stress (e.g., time and temperature) on the ability to determine a SARS-CoV-2 simulant, i.e., HCoV-OC43, collected on 37 mm 1 μm pore-size laminated PTFE filter in a conductive plastic cassette (Vire-Pore; Environmental Express, Inc.). The laminated PTFE filter used in this work does not include an attached non-PTFE backing. Backings are characteristic of other PTFE filters to prevent the thin filters curling up. This filter was specifically chosen to ensure that any backing material does not interfere with any analytical procedure. Extrapolation of these results to alternative filters should be assessed carefully before use.

3. MATERIALS AND METHODS

3.1. Preparation of Human Coronavirus OC43 Inoculum

The human coronavirus OC43 (HCoV-OC43) was prepared according to published protocols (DeDiego et al. 2007). Briefly, Vero E6 (African green monkey kidney; ATCC CRL-1586) cells were cultured at 33°C in Eagle’s minimum essential medium (MEM; ThermoFisher Scientific Inc., Pittsburgh, PA, USA) supplemented with 10% fetal bovine serum (FBS; ThermoFisher Scientific Inc.). HCoV-OC43 (ATCC VR-1558) was added to the Vero E6 cell culture at the indicated MOI (multiplicity of infection) and incubated for adsorption at 33°C for one hour. Unbound virus was removed by washing cells three times with ice-cold phosphate-buffered saline solution (PBS; Sigma-Aldrich Co., St. Louis, MO, USA), followed by introducing fresh MEM to the cells. Media from infected cultures was then harvested at various times and centrifuged at 1500 rpm (536 x g) for 15 mins at 4°C to remove any cell debris. The supernatant that contained the HCoV-OC43 in MEM, including any traces of the Vero E6 cells and by-products not removed by centrifugation, were transferred to a 50 ml conical tube. Titers were determined from the supernatant by plaque assay using Vero E6 cells. HCoV-OC43 is a BSL2+ virus (ATCC), and for safety reasons and to adhere to Rutgers biosafety protocols, the virus was inactivated by heating its suspension in a water bath at 56°C for 30 mins and kept frozen at −80°C until aerosolization experiments. The absence of plaques for the inactivated virus was confirmed by plaque assay on Vero E6 cells.

3.2. Experimental Setup

The test system used to aerosolize, measure, and sample viral aerosol is based on our design described earlier (Han et al. 2017; Han et al. 2018) (Figure 1). The test system consists of a flow controller, a particle generator, an air-particle mixing element, a flow straightener, a test chamber, and a particle monitor (Han et al. 2017). The system is housed in a Class II Biosafety cabinet (NUAIRE Inc., Plymouth, MN, USA). The liquid suspension with the inactivated virus was aerosolized using a six-jet Collison nebulizer with a polycarbonate jar and operated at a flow rate, QA= 5 L/min (pressure of 19 psig). As per our earlier research, using a polycarbonate jar and low aerosolization pressure minimizes damage to biological particles (Zhen et al. 2013).

Figure 1.

Figure 1.

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Schematic of the experimental setup.

The aerosolized viral particles were combined with a dry airflow, Qd (5 L/min) (Zhen et al. 2013; Han et al. 2015). The flow stream was passed through a 2-mCi Po-210 charge neutralizer (Amstat Industries Inc., Glenview, IL, USA) to reduce aerosolization-imparted particle charges to Boltzmann charge equilibrium. A HEPA-filtered dilution airflow, QD (60 L/min), provided by an in-house compressor, dried and diluted the particle stream. A well-mixed flow stream was then passed through a flow straightener (honeycomb). The filter cassette with the filter being tested was positioned six duct diameters downstream of the exit of the flow straightener to provide a uniform cross-sectional particle profile (Han et al. 2018). The concentration of airborne particles (#/L) was monitored by an SMPS 3080 + CPC 3776 system (both TSI Inc., Shoreview, MN, USA) and GRIMM MiniWRAS 1371 (GRIMM Aerosol Technology, Ainring, Germany) that were connected to an isokinetic probe (Apex Instruments Inc., Fuquay-Varina, NC) via a flow splitter (TSI Inc.).

Temperature and relative humidity have been documented to affect the nucleic acid integrity of airborne viruses after sampling (Verreault et al., 2008). For example, Ijaz et al. (1987) reported up to 90% recovery in virus infectivity of Human coronavirus 229E at the mid-range relative humidity (50 ± 5%). Since one of our main study goals was to understand variations in virus recovery specific to sampling flow rate and sampling time, experiments were performed at stable environmental conditions with room temperatures within 20°C – 22.2°C (68 – 72°F) range and mid-range relative humidity of 40-60%.

3.3. Aerosolization of the HCoV-OC43 Virus from MEM

We aerosolized the HCoV-OC43 directly from the growth medium, and the resulting aerosol contained OC43+ MEM + traces of Vero E6 cells and by-products. The aerosol was measured by the instruments mentioned above. Pan et al. (2019a) used a similar approach and reported the size and volume distribution of MS2 bacteriophage aerosolized from beef extract solution. Size distribution of particles (viruses in this case) aerosolized from salt-containing solutions are dominated by the salt and other non-virus components of the aerosol, but measuring that aerosol serves as a quality control feature informing us about the stability of aerosol production. In addition, it indirectly conveys information about the concentration of viral particles in the air.

3.4. Sampling Flow Rates, Pressure Drops, and Pumps

The aerosolized HCoV-OC43 virus was sampled using Vira-Pore conductive polypropylene cassettes equipped with 1 μm pore-size 37mm ZePore ™ laminated PTFE filters (Environmental Express, Inc., Ocala, Florida, USA) used in an “open-face” configuration. Conductive cassettes are preferred for fine particle sampling due to lower sampling losses (Baron 2003). Two different sampling flow rates were used:

  1. 10 L/min, which with a support pad included leads to a pressure drop across the sampler of 17 inches w.g. (4.23 kPa), which allows the use of certain personal pumps, in addition to vacuum pumps.

  2. 3 L/min, which with a support pad included leads to a pressure drop across the sampler less than 5.5 inches w.g. (1.37 kPa), which is compatible with many personal sampling pumps.

The flow rate was verified using a calibrated mass flowmeter (TSI Inc.). The sampling duration varied between 10 and 60 min depending on the experiments described in Sections 3.5.4 and 3.5.5.

3.5. Test Parameters

3.5.1. Testing of Elution Method for Viral Particles Spiked onto Filters

Filters were placed in sterile 60 mm Petri dishes (Fisher Scientific Inc., Boston, MA, USA), and 500 μL of the virus suspension was added onto the filters in small droplets (Figure 2). The filters were then placed in an incubator at 25°C (room temperature) for approximately four hours or until the liquid evaporated. The viral RNA in the control sample was not subjected to any stress before its extraction and was used to determine the recovery of the virus after elution. PrimeStore® MTM media (Longhorn Vaccines and Diagnostics™, San Antonio, TX, USA) was the elution liquid of choice since it is known to inactivate viruses and act as an RNA stabilization agent (Daum et al. 2011; Daum et al. 2014; Zar et al. 2016). Once the suspension dried out, the viral particles were eluted using the following two methods:

Figure 2:

Figure 2:

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Spiked and dry filters with HCoV-OC43 virus stock solution.

  1. Shaker method: The filters were placed face down in a 60 mm petri dish (Fisher Scientific Inc.) with 700 μL of PrimeStore® MTM media (Longhorn Vaccines and Diagnostics™ Inc.) and sealed with parafilm tape (Fisher Scientific Inc.). The petri dish was securely taped onto an orbital shaker for 20 mins at 360 rpm (Figure 3). The shaker method was published earlier (Booth et al., 2005, Myatt et al., 2003 and 2004).

  2. Vortex method: The filters were rolled inwards and placed in a 2 mL microcentrifuge tube (Fisher Scientific Inc.) with 1 mL of PrimeStore® MTM media (Longhorn Vaccines and Diagnostics™, Inc.) and vortexed three times for 10 seconds each (Figure 4). The vortex method was similar to other published elution techniques (Coleman et al., 2018, Fabian et al., 2009, Jonges et al., 2015, Tseng & Li, 2005).

Figure 3:

Figure 3:

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Elution of virus spiked onto filters using the shaker method.

Figure 4:

Figure 4:

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Elution of virus spiked onto filters using the vortex method.

3.5.2. Testing of Elution Method for Viral Particles Collected onto Filters

The viral particles were aerosolized as described above (Figure 1) and then sampled on filters for 10 mins at 10 L/min. Next, triplicate samples of the captured virus were eluted using the two methods described in Section 2.4.1. PrimeStore® MTM (Longhorn Vaccines and Diagnostics™ Inc.) was used as the elution liquid, and the eluted samples were immediately placed on ice before RNA extraction.

3.5.3. Testing of Support Pads for RNA Interference

Sampling cassettes are frequently used with support pads to minimize pressure drop. Separate support pads will not interfere with the analysis, although they might transfer any residual RNA to the filters since they come in contact with filters. Thus, cellulose and porous plastic support pads (PW37100 and PFSP37; Environmental Express, Inc.) 37 mm in diameter were tested for RNA interference with the collected virus samples. Cellulose pads were folded into halves and placed in 5 ml sterile tubes (Fisher Scientific Inc.) with 2 ml of PrimeStore® MTM (Longhorn Vaccines and Diagnostics™ Inc.). The tubes were vortexed thrice for 10 seconds each. When testing porous plastic pads, each side of the pad was rinsed with 2 ml of sterile RNA-free water (Fisher Scientific Inc.) and dried. The rinsed-off and vortexed liquids, including controls (e.g., water and MTM), were immediately quantified for their total viral RNA concentration.

3.5.4. Impact of Sampling Stress on Airborne Virus Recovery

To evaluate the impact of sampling stress on the recovery of airborne virus captured on ZePore ™ filters, the RNA yields were compared between experiments with no added stress (filters analyzed immediately after sampling) and with added stress, where HEPA-filtered air was passed through the filter containing viral particles either at 3 or 10 L/min for a certain duration. The six experiments are described in detail in Table 1.

Table 1.

Experiments designed to study the impact of sampling stress on airborne virus recovery

Experiment No. Sampling Flow Rate, L/min Sampling Time Sampled Air Volume, L Added Stress Total air volume passed through a filter, L
1 10 10 mins 100 No added stress 100
2 10 10 mins 100 Clean airstream for 1 hour 700
3 10 1 hour 600 No added stress 600
4 3 1 hour 180 No added stress 180
5 3 1 hour 180 Clean airstream for 5 hours 1080
6 3 1 hour 180 Clean airstream for 15 hours 2880
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Each experiment was performed at least in triplicate. In addition to eluted virus samples, positive (fresh viral suspension) and negative (elution liquid and MEM media) controls were analyzed. The collected viral particles were eluted into the PrimeStore® MTM media (Longhorn Vaccines and Diagnostics™, Inc.) immediately after sampling, and the liquid was quantified for its total RNA concentration.

3.5.5. Impact of Storage Stress on Airborne Virus Recovery

The recovery of the virus from the filter was evaluated for specific short-term storage periods and temperature conditions. The RNA yields were compared between experiments with sampling and immediate analysis (no added stress) versus experiments with sampling followed by storage at different temperatures and durations. The following three experiments were performed (details in Table 2):

Table 2.

Experiments designed to study the impact of sampling storage on airborne virus recovery

Experiment No. Sampling Flow Rate, L/min Sampling Time Sampled Air Volume, L Storage Conditions
1 3 1 hour 180 No storage
2 3 1 hour 180 2-day storage at 25°C
3 10 10 mins 100 No storage
4 10 10 mins 100 1-week storage at 4°C
5 10 10 mins 100 1-week storage at 25°C
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  1. Sampling of the aerosolized virus at 3 L/min for 1 hour (180 L air volume) and storing the filter at 25°C (room temperature) for two days (48 hours) to simulate the time commonly needed to transport unrefrigerated filters from a sampling site to a laboratory.

  2. Sampling of the aerosolized virus at 10 L/min for 10 mins (100 L air volume) and storing the filter at 4°C for seven days to simulate storing filters for a short term in refrigerated conditions prior to analysis.

  3. Sampling of the aerosolized virus at 10 L/min for 10 mins (100 L air volume) and storing the filter at 25°C (room temperature) for seven days to simulate storing filters for a short term at room temperature prior to analysis.

Each experiment was performed at least in triplicate, and the eluted samples and positive (fresh viral suspension) and negative (elution liquid and MEM) controls were analyzed. After the specified storage condition, the collected viral particles were eluted into the PrimeStore® MTM media (Longhorn Vaccines and Diagnostics™, Inc.), and their total RNA concentration was quantified.

3.6. Viral Quantification Method

The QIAamp Viral RNA Mini Kit (Qiagen Co., Germantown, MD) was used for extraction, and the manufacturer’s protocol was optimized to obtain a higher RNA yield and quality. This protocol uses 5.6 μg carrier RNA for each sample. The total RNA mass concentration measured in samples is the sum of mass concentrations of the eluted or spiked viral RNA from HCoV-OC43 and the carrier RNA. To determine the actual HCoV-OC43 RNA concentration, the carrier RNA mass concentration was determined in the blank filter sample and then subtracted from the total RNA mass concentration of samples. By our estimates, the kit has a yield of 35-40% compared to the standard trizol-chloroform extraction procedure (data not shown). Similar results for this kit were published by Fabian et al., 2009, where the QIAmp kit recovered 2.5x less RNA than the trizol-chloroform extraction procedure. Nonetheless, we chose to use the QIAmp kit since it contains no hazardous materials (e.g., chloroform). In addition, it takes less time to process multiple samples simultaneously than the phenol-chloroform extraction procedure.

Sixty microliters of RNA were extracted from the eluted samples and immediately put on ice. The extracted RNA samples were quantified using the Nanodrop 1000 (ThermoFisher Scientific Inc.; Rutgers SEBS Core Facilities) in ng/μL. The NanoDrop measures nucleic acid concentration by absorbance using a 2 μL RNA sample and assesses the extracted RNA’s purity using the A260/A280 ratio (Desjardins and Conklin 2010).

3.7. Recovery Metrics

3.7.1. Relative Recovery of Captured Virus Normalized to the Sampled Air Volume

The results were expressed as a ratio of recovered viral RNA concentration relative to viral RNA concentration in the initial suspension placed in the nebulizer and normalized to the sampled air volume (RRmass). This method for calculating the relative recovery of RNA from phage aerosols was published earlier (Gendron et al. 2010). RRmass was calculated using the following formula:

RRmass(L−1)=MassconcentrationofviralRNAelutedfromfilter(ngml)xmultiplicationfactorof1000MassconcentrationofviralRNAintheinitialsample(ngml)∗Sampledairvolume(L) (1)

3.7.2. Total Viral Mass Normalized to the Number of Virus Aerosol Particles

The virus recovery data were also presented in terms of total viral mass eluted from filters and normalized to the number of virus aerosol particles (TVMaerosol). The total viral mass was calculated based on the viral mass concentration measured by Nanodrop 1000 (ThermoFisher Scientific Inc.) and the final volume of liquid used for elution, i.e., 1 ml for the vortex elution method. The GRIMM MiniWRAS 1371 (GRIMM Aerosol Technology Inc.) measures airborne particle size distribution every six seconds. Due to this ease of use, it was used to determine the total number of virus aerosol particles from 27 to 680 nm. TVMaerosol was calculated using the following formula

TVMaerosol(mass,ag#)=MassconcentrationofviralRNAelutedfromfilter(ngml)∗Totalvolumeofliquidusedforelution(ml)AirbornenumberconcentrationbasedonMiniWRAS(#L)∗Sampledairvolume(L) (2)

Where ag stands for attogram, 10−18 g

3.8. Data Analysis

SPSS v27.0 (IBM, Armonk, NY, USA) and OriginPro 2019 (OriginLab, Northampton, MA, USA) were used to analyze and illustrate the data, respectively. Grayscale colors with patterns available in OriginPro were chosen for visualizing the data in the figures. The data are presented as an average and standard deviation of the repeats for each experiment. The statistically significant differences among three or more groups were determined by one-way ANOVA, followed by the Tukey’s-b post-hoc analysis to identify pairs with statistically significant differences. The statistical difference of the mean between any two groups was determined by the independent samples t-test. Groups with p-values of less than 0.05 were considered significantly different, and groups with p-values of less than 0.1 were considered to be trending but not significantly different.

3.9. Biosafety Protocols

Appropriate biosafety protocols involved in the transport, handling, and aerosolization of the virus were approved by the Rutgers Environmental, Health, and Safety Service. All experiments were conducted in a Class II Biosafety cabinet (NUAIRE Inc.).

4. RESULTS AND DISCUSSION

4.1. Size Distribution of the HCoV-OC43 Aerosol

The electrical mobility size distribution of HCoV-OC43 aerosol measured by SMPS 3080 and CPC 3776 system (both TSI Inc.) and MiniWRAS (Grimm Technologies Inc.) is shown in Fig. 5. The SPMS registered aerosol mode diameter of ~40 nm, while the mode diameter by the MiniWRAS was ~60 nm. While both instruments size and count particles based on their electrical mobility, their principle of particle detection and mathematical algorithms are different. Still, given their differences, the two instruments could be considered in reasonable agreement. A more detailed discussion regarding the reasons for the observed differences is beyond the scope of this paper.

Figure 5:

Figure 5:

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Size distribution of HCoV-OC43 virus aerosolized from MEM suspension and measured by SMPS 3080 and CPC 3776 system (both TSI Inc.) and MiniWRAS (Grimm Technologies Inc.). The presented data points and the error bars (one standard deviation) are based on a 10 min measurement.

4.2. Method to Elute Spiked and Airborne Viral Particles from Filters

The recovery of the viral particles spiked onto filters varied depending on the elution method. For example, vortex and shaker methods recovered 84% and 54% of the virus particles, respectively. A similar result was shown by Jonges et al. (2015), where vortexing three times for 10 sec each recovered ~2x higher concentrations of influenza virus than a bench rocker operated for 30 minutes.

For the filter samples with airborne viral loading of 10 mins at 10 L/min, the RRmass for the vortex and shaker methods were 6.7 and 3.4, respectively. Based on these results, all subsequent filters were processed by the vortex method prior to RNA extraction and quantification by Nanodrop 1000 (ThermoFisher Scientific Inc.). Other studies using filter sampling have also applied a similar vortex method for the recovery of airborne viruses, such as for the recovery of influenza virus (Fabian et al. 2009), bacteriophages used as surrogates for mammalian viruses (Tseng and Li 2005), and respiratory viruses such as adenovirus and respiratory syncytial virus (Coleman et al. 2018).

4.3. RNA Interference by Support Pads

Based on experiments with seven cellulose pads and three porous plastic pads, the average RNA concentration measured in the rinsate did not differ significantly from the control samples (p > 0.05). Yet, in two samples rinsed off from cellulose pads, the RNA concentrations were higher than in the control samples.

Furthermore, previous experiments with water and MTM (Longhorn Vaccines and Diagnostics™ Inc.) spiked with carrier RNA showed no difference in the resulting RNA concentration once the samples had been processed. So, either one of the two liquids could be used to rinse off the porous plastic pads. Due to the cost and limited availability of MTM (Longhorn Vaccines and Diagnostics™ Inc.), we chose to rinse off the porous plastic pads with sterile RNA-free water (Fisher Scientific Inc.).

4.4. The Effect of Sampling Stress on the Recovery of Captured Airborne Virus

4.4.1. Relative Recovery Normalized to the Sampled Air Volume

The six experiments designed to evaluate the effect of sampling stress are presented in Table 1, and the obtained RR mass of the airborne virus collected on filters are presented in Figure 6. For the filters sampled at 10 L/min for 10 mins (Column 1), 10 L/min for 10 mins along with added stress of clean air for 1 hour (Column 2), and 10 L/min for 1 hour (Column 3), the RRmass were 6.7 L−1 (± 1.3), 5.0 L−1 (± 1.6), and 5.4 L−1 (± 3.3), respectively.

Figure 6:

Figure 6:

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The effects of sampling stress on relative Recovery (RRmass; L−1) of airborne HCoV-OC43 sampled on ZePore™ filters (Environmental Express Inc.). Columns 1, 2, 3, 4, 5, and 6 represent sampling at 10 L/min for 10 mins (100 L), 10 L/min for 10 mins (100 L), followed by exposure to clean airstream for 1 hour, 10 L/min for 1 hour (600 L), 3 L/min for 1 hour (180 L), 3 L/min for 1 hour (180 L) followed by exposure to clean airstream for 5 hours, and 3 L/min for 1 hour (180 L) followed by exposure to clean airstream for 15 hours, respectively. The six groups were not significantly different from each other, with a p-value of 0.805.

Similar results were obtained when samples were collected using a lower flow rate of 3 L/min. For the filters sampled at 3 L/min for 1 hour (Column 4), 3 L/min for 1 hour along with added stress of clean air for 5 hours (Column 5), and 3 L/min for 1 hour along with added stress of clean air for 15 hours (Column 6), the RRmass were 5.5 L−1 (± 2.3), 4.8 L−1 (± 2.0), and 4.2 L−1 (± 1.8), respectively.

These results show that the RRmass was similar to each other after sampling and immediate analysis for air volumes of 100 L, 100 L followed by exposure to clean airstream for 1 hour, 600 L, 180 L, and 180 L followed by exposure to clean airstream for 5 hours and 15 hours; the difference between each RRmass was not statistically significant (p = 0.805).

4.4.2. Total Viral Mass Normalized to the Virus Aerosol Particle Number

The TVMaerosol values for the six experiments are presented in Figure 7. For the filters sampled at 10 L/min for 10 mins (Column 1), 10 L/min for 10 mins along with added stress of clean air for 1 hour (Column 2), and 10 L/min for 1 hour (Column 3), the TVMaerosol were 134.9 ag/# (± 74.9), 127.5 ag/# (± 40.9), and 86.3 ag/# (± 16.3), respectively.

Figure 7:

Figure 7:

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The effect of sampling stress on the recovery of total viral RNA when samples were collected on ZePore™ filters (Environmental Express Inc.). The results are expressed as the total viral mass of HCoV-OC43 normalized to the viral aerosol particle number (TVMaerosol; attogram/# ). Columns 1, 2, 3, 4, 5, and 6 represent sampling at 10 L/min for 10 mins (100 L), 10 L/min for 10 mins (100 L), followed by exposure to clean airstream for 1 hour, 10 L/min for 1 hour (600 L), 3 L/min for 1 hour (180 L), 3 L/min for 1 hour (180 L) followed by exposure to clean airstream for 5 hours, and 3 L/min for 1 hour (180 L) followed by exposure to clean airstream for 15 hours, respectively. The six groups were not significantly different from each other, with a p-value of 0.466.

Similar results were also obtained when filter samples were collected at a lower flow rate of 3 L/min. For the filters sampled at 3 L/min for 1 hour (Column 4), 3 L/min for 1 hour along with added stress of clean air for 5 hours (Column 5), and 3 L/min for 1 hour along with added stress of clean air for 15 hours (Column 6), the TVMaerosol were 157.8 ag/# (± 66.4), 147.0 ag/# (± 64.6), and 97.4 ag/# (± 42.7), respectively.

These results show that the TVMaerosol for the six different sampling scenarios of HCoV-OC43 were not significantly different (p = 0.466).

4.5. The Effect of Storage Stress on the Recovery of Sampled Airborne Virus

4.5.1. Relative Recovery Normalized to the Sampled Air Volume

The experimental design to evaluate the effect of storage stress is presented in Table 2, and the obtained RR mass of the airborne virus collected on filters is presented in Figure 8. The RRmass of airborne virus sampled at 3 L/min for 1 hr with two-day storage at 25°C was 3.7 L−1 (± 1.4) (Column 2). Although this RRmass was 1.49x lower than after immediate analysis with no storage (Column 1), the difference was not statistically significant (p = 0.24).

Figure 8:

Figure 8:

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The effect of storage stress on relative Recovery (RRmass; L−1) of airborne HCoV-OC43 sampled on ZePore™ filters (Environmental Express Inc.). Columns 1 and 2 represent sampling at 3 L/min for 1 hour (180 L air volume) with no storage and 2-day storage at 25°C, respectively. Columns 3, 4, and 5 represent sampling at 10 L/min for 10 mins (100 L air volume) with no storage, 1-week storage at 4°C, and 1-week storage at 25°C, respectively. The five groups were not significantly different from each other but trending with a p-value of 0.059. When pairwise comparisons within this group of five were considered, 10 L/min for 10 mins (100 L air volume) with no storage and 1-week storage at 25°C were significantly different from each other (p <0.05).

For the filters sampled at 10 L/min for 10 min (100 L air volume) and with 7-day storage at 4°C (Column 4) and 25°C (room temperature; Column 5), the RRmass were 5.5 L−1 (± 3.1) and 1.7 L−1 (± 1.0), respectively. Thus, the average RRmass after 7-day storage was about 3.2x lower for room temperature storage than for refrigerator storage. As shown in Figure 8, when comparing the RRmass of three data sets for the same sampling condition of 10 L/min for 10 min (100 L air volume) but different storage stresses, i.e., 7-day storage at 25°C, 7-day storage at 4°C, and no storage (Column 3), the statistical difference was trending, but not significantly different with p = 0.053.

Overall, when all five sampling scenarios in Figure 8 were considered, the statistical difference in RRmass was trending but not significantly different with p = 0.059. When pairwise comparisons within this group of five were considered, only sampling for 10 mins at 10 L/min (100 L air volume) with no storage and 1-week storage at 25°C were significantly different from each other (p <0.05).

4.5.2. Total Viral Mass Normalized to Virus Aerosol Particle Number

The effect of storage stress by evaluating the total viral mass normalized to the virus aerosol particle number (TVMaerosol; attogram/#) is presented in Figure 9. The TVMaerosol for the filters sampled at 3 L/min for 60 min with two days of storage at 25°C was 79.8 ag/# (± 29.9) (Column 2). The TVMaerosol was 1.98x lower than for immediate analysis with no storage (Column 1), and the statistical difference was trending but not significantly different with p = 0.076.

Figure 9:

Figure 9:

Open in a new tab

The effects of storage stress on total viral mass normalized to the viral aerosol particle number (TVMaerosol; attogram/# ) sampled on ZePore™ filters (Environmental Express Inc.). Columns 1 and 2 represent sampling at 3 L/min for 1 hour (180 L air volume) with no storage and 2-day storage at 25°C, respectively. Columns 3, 4, and 5 represent sampling at 10 L/min for 10 mins (100 L air volume) with no storage, 1-week storage at 4°C, and 1-week storage at 25°C, respectively. The five groups were not significantly different from each other but trending with a p-value of 0.091.

The TVMaerosol for the filters sampled at 10 L/min for 10 min along with 7-day storage at 4°C (Column 4) and 25°C (Column 5) were 118.6 ag/# (± 67.4) and 36.1 ag/# (± 22.2), respectively. Similar to the RRmass results, the average TVMaerosol for 7-day storage was ~3.3x lower for room temperature storage than for refrigerator storage. Even though there was a difference in the average TVMaerosol of three data sets for the same sampling conditions of 10 L/min for 10 min with different storage stresses, i.e., 7-day storage at 4°C, 7-day storage at 25°C, and no storage (Column 3), it was not statistically significant (p = 0.184).

Overall, when all five sampling scenarios in Figure 9 were considered, the statistical difference in TVMaerosol was trending, but not significantly different with p = 0.091, with the two lowest TVMaerosol measured for storing filters at room temperatures.

5. LIMITATIONS AND FUTURE DIRECTIONS

There are a few limitations of the study. Firstly, one type of PTFE filter (ZePore™ filter) was studied using the surrogate of the SARS-CoV-2 virus. Nevertheless, the main advantage of using this filter is the possibility of long-term sampling. Overall, our data showed no significant difference (p > 0.05) in sampling stress for sampling of up to 16 hours and 2880 L of total air volume passed through the filter. Furthermore, the results are expressed in relative terms, and so any potential losses of virus particles during sampling and elution will be equivalent for all the sampling scenarios. Other filters such as gelatin filters can completely dissolve in warm media and have been used to study influenza viruses (Fabian et al. 2009; Ka et al. 2018; Kormuth et al. 2019) and recently the SARS-CoV-2 virus (van Doremalen et al. 2020; Rodríguez et al. 2021). However, gelatin filters can only be used for shorter sampling periods (i.e., 15 – 20 mins) and can desiccate and crack with longer sampling periods (Burton et al. 2005; Lindsley et al. 2017). Additionally, Fabian et al. (2009) reported similar collection and recovery efficiencies of total influenza virus RNA between PTFE and gelatin filters.

Secondly, the recovery metrics based on RNA concentrations and PCR analysis are common techniques used for total viral quantification. This technique eliminates the need to preserve viral viability. However, when using PCR, information on viral infectivity is lost and analysis on viral transmission between individuals becomes difficult (Lindsley et al. 2017). Future testing can include culturing the virus in a cell culture system compatible with the virus (Lednicky et al. 2020).

Thirdly, the storage stress experiments were designed to simulate short-term storage scenarios, where samples are collected in the field and then shipped to laboratories for analysis, and the analysis is performed within a week from field sampling. Given the immediate need for monitoring and contact tracing for any potential exposures to the SARS-CoV-2 virus, our results can guide field personnel and laboratories that need short-term storage and a quick turnaround time. Furthermore, storing filters used to sample airborne viruses at 4°C instead of −20°C or −80°C avoids the freeze/thaw cycle and reduces the possibility of RNA degradation (Granados et al. 2017). One of the challenges of virus sampling and detection is the prevalence of low viral concentrations in the air (Pan et al. 2019b). Viral samples with low concentrations have been reported to have lower RNA stability over time than highly concentrated samples (Granados et al. 2017). Therefore, our recommendations reduce any additional stress to viral RNA due to short-term storage. Moreover, storage recommendations similar to our study were reported by Damen et al. (1998), where stable loads of Hepatitis C Virus (HCV) RNA in serum were detected when stored up to one week at 4°C and two days at 30°C. Chin et al. (2020) analyzed the stability of SARS-CoV-2 at different temperatures and found that the virus is stable at 4°C up to 14 days with less than one log-unit reduction of the infectious titer. However, our study does not focus on long-term storage scenarios at sub-zero freezing temperatures, and this aspect could be explored in future studies.

6. CONCLUSIONS AND RECOMMENDATIONS

HCoV-OC43, a surrogate for the novel SARS-CoV-2 virus, was aerosolized from a growth medium, and the resulting aerosol had a mode diameter of 40-60 nm as per SMPS + CPC system (TSI Inc.) and MiniWRAS (Grimm Technologies Inc.). The use of HCoV-OC43 aerosol as a surrogate for airborne SARS-CoV-2 adds to the novelty of the study. The vortex method of elution yielded higher RNA concentrations than the shaker method for both virus samples spiked on the filter and airborne virus samples collected on the filter used in this study, which is a 1-micron pore-size, laminated PTFE with no non-PTFE support. For the sampling cassettes, we recommend using porous plastic support pads to minimize pressure drop during sampling. Cellulose pads are not recommended because of potential contamination and RNA interference. In addition, the filter holder (cassette) should be conductive plastic to minimize the deposition of particles on the walls of the cassette. The combination of laminated PTFE filter, polypropylene support pad, and the conductive plastic cassette is commercially available (Vira-Pore, Environmental Express, Inc.).

Short and long-term sampling was conducted to analyze the impact of sampling and storage stress on the airborne virus. When HCoV-OC43 collected on the filter was exposed to different sampling stress, there was no statistical difference between low (3 L/min) and high (10 L/min) sampling flow rates and sampling duration between 10 min and 60 min.

For the experiment designed to simulate shipping of the filters in 2 days, the recovery metrics of the virus for unrefrigerated room temperature conditions (25°C) were up to ~2x lower compared to immediate processing after sampling. Consequently, the filters are recommended to be shipped in refrigerated containers or using ice packs to maintain lower temperatures.

For the experiments designed to simulate storing of the filter in a laboratory for a short period before elution and analysis, the recovery metrics for the 7-day storage at unrefrigerated room temperature (25°C) were lower than for the 7-day storage in a refrigerator (4°C) and immediate processing of filters. Therefore, it is recommended to store filter samples with viruses in a laboratory refrigerator (4°C) prior to analysis if the immediate analysis is not feasible.

Overall, we conclude that for both qualitative and quantitative analyses of the airborne SARS-CoV-2 virus surrogate captured by the PTFE filters used in this study, filters are recommended to be shipped in an insulated cold container within a week from sampling or stored short-term in a laboratory refrigerator (4°C) before analyses. In addition, with the broad use of PTFE filters to capture airborne viruses worldwide, researchers can apply our recommendations to other PTFE filter types to decrease the impact of sampling and storage stress on airborne virus recovery.

Financial Support:

We thank Antylia Scientific Company LLC for funding the study.

Conflict of Interest:

Dr. Martin Harper is employed by Environmental Express, an Antylia Scientific company, and receives funding as Commissioning Editor for JOEH.

Role of Funder:

The sponsor agreed to the publication of this work.

Footnotes

Public Accessibility: Study data are available from the corresponding author upon request.

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