In the current global geopolitical environment, the threat of a terrorist attack on the US and international community has never been greater. A biological threat of great concern to public safety is the use of an aerosolized pathogenic micro-organism that can be spread in the air as happened with the anthrax attack of 2001 (Zink, 2011; Menrath et al., 2014). In addition, an individual infected with a deadly pathogen can transmit the pathogen at a high human density place or event. Some highly contagious and fatal biological agents that are considered as bio-terror agents include Bacillus anthracis, Yersinia pestis, small pox, or Marburg viruses and other emerging pathogens (Pohanka and Kuca, 2010; Menrath et al., 2014).

Besides the intentional release of bioaerosols, natural outbreaks of infectious diseases may occur without warning. Outbreaks including Severe Acute Respiratory Syndrome (SARS) in 2002–2004 (Christian et al., 2004), continuously evolving influenza virus strains (Khanna et al., 2008) and recent infections with Middle East respiratory syndrome coronavirus (MERS-CoV; Hartl, 2013) underscore that such a natural threat is a real possibility. Early detection of the pathogen can result in prompt intervention and treatment of exposed individuals as well as confine the secondary spread. Because of the high global mobility of people, the failure to detect an intentional or natural outbreak of a contagious disease could result in its rapid spread with catastrophic consequences. In order to effectively safeguard against such threats, it is critical to continuously monitor the air for the presence of pathogenic biological agents, particularly at high risk events or places such as airports, malls, subway stations, cruise ships, aircraft, theaters, stadiums, or schools. Such an approach requires an advanced system that can continuously sample the ambient air for pathogen detection.

Following the acquisition of an air sample, many different approaches including genome detection (Pyankov et al., 2007; Goransson et al., 2012; Usachev and Agranovski, 2012), immune (McBride et al., 2003; Rider et al., 2003; Skottrup et al., 2008), optical (Pan et al., 2003; Sengupta et al., 2007), or other biochemical assays (Hagleitner et al., 2001; Ho, 2002) have been employed for specific detection of a large selection of pathogens including bacteria, fungi, virus, or toxins. Since nucleic acids are the absolute biomarkers, quantitative polymerase chain reaction (qPCR) or quantitative reverse transcriptase-PCR (qRT-PCR) is strongly established as the method of choice for accurate, sensitive and specific identification of a number of pathogens in the same sample (Oppliger et al., 2011).

The Airborne Sample Analysis Platform (ASAP) 2800 (Thermo Scientific, USA) bioaerosol sampler provides an effective means of collecting representative samples of air for subsequent laboratory analysis. At a flow rate of 200 l/min, this sampler pulls in the air which impacts on a piece of polyurethane foam (PUF) inside a cassette called Integrated Bio Aerosol Sampling System (iBASS) cartridge (Figure 1). In this study we investigated the efficiency of the ASAP system to acquire an aerosolized viral pathogen and detect it using a Taqman qPCR. For testing, we aerosolized an innocuous replication-defective bovine adenovirus serotype 3 (BAdV3) in a controlled laboratory environment. The ASAP system successfully trapped the virus-loaded aerosols which were subsequently detected by qPCR.

Over the last several decades, continuous air sampling coupled with rapid and accurate detection of potential pathogens in bioaerosols has attracted much attention. The ASAP bioaerosol sampler has been tested to sample bacteria and spores (Goransson et al., 2012) or metalliferous airborne particles (Moreno et al., 2015) under field conditions. However, the ASAP sampler had not yet been tested for collection of viral aerosols. In this study we investigated the efficacy of the ASAP bioaerosol sampler to collect and trap surrogate virus aerosols from the air and detect them using suitable assays. We demonstrated that in controlled laboratory conditions, the ASAP sampler can efficiently trap surrogate BAd-ΔE1E3 viral aerosols that can be extracted from the PUFs and identified using qPCR.

The purpose of bioaerosol sampling is to detect a biological agent release so that the impact of a biological agent attack can be reduced by executing appropriate responses with minimal delay. However, the conventional time-consuming laboratory methods involving culturing of microorganisms can take days to identify the pathogen. Molecular biology techniques such as qPCR are rapid, specific and sensitive enough to identify the genus, species or strain of the pathogen. These molecular assays can also be multiplexed to monitor more than one bio-threat agent at a time. As qPCR assays are quantitative, they can provide additional valuable information regarding the concentration of pathogens in the ambient air. We successfully detected the bioaerosols containing as low as 2 × 10⁵ genome copies equivalent (5 × 10³ p.f.u.) of BAd-ΔE1E3. The technology of airborne pathogen detection is still in the trial stage with no approved device in practical application yet. Unfortunately, there are no set standards for the sensitivity of airborne pathogen detection. However, the detection of surrogate virus, released from a distance of 10 ft (305 cm), and at a concentration as low as 2 × 10⁵ genome copies equivalent (5 × 10³ p.f.u.) in a room the size of 972 cubic ft (27.5 cubic meters) is quite sensitive by many standards.

The majority of the currently available air sampling platforms are based on either impaction onto agar, impingement into liquid, or dry impaction (Kesavan and Sagripanti, 2015), and each has its limitations. The direct collection of microbes on agar is limited since not all species can be cultured and others have distinct nutrient requirements. The agar surface can also get saturated in areas with high concentrations of microbes, and it cannot be used for viruses.

For viruses and other viable microorganisms, the collection of aerosols in liquid media/buffer is a preferred method. However, the high rate of sample collection usually results in violent bubbling of the collection fluid resulting in considerable loss of collection fluid within short time (Agranovski et al., 2004). Therefore, samplers based on liquid impinging or bubbling usually operate at lower speeds. Some advancement has been made to address these problems.

In the dry method (this study), the high volume of air impacting on the PUF results in a strong desiccation effect during sampling which dramatically diminishes the viability of most microorganisms. The inefficiency of dry impaction to collect viable particles is often considered a limitation; however, this method allows high rates of sample collection (>200 l/min) enabling rapid analysis of a larger sample volume from the environment.

The advantage of molecular techniques is that the genetic material can be detected even from inactive microorganisms. The specific detection of the genome of potentially dangerous microorganisms in the aerosol samples is evidence of a direct threat serious enough to raise an alarm and initiate appropriate measures. In this study we use only a DNA virus to test the efficacy of ASAP sampler. The iBASS cartridge is removed after every 4–8 h of operation for analysis of the microorganisms collected on the PUF, and during this time the genetic material, even from the RNA viruses, is expected to remain stable inside the capsid/shell of the inactive virus. It has been shown that phage PR772 and possibly other viruses as well, lost their infectivity before the genome degradation (Turgeon et al., 2014). Additionally, it has been demonstrated that the overall recovery of airborne viruses should be estimated based on the detection of genetic material rather than on viral infectivity (Verreault et al., 2010; Turgeon et al., 2014). The use of molecular biological methods is more likely to avoid false negative results than infectivity assays for the detection of airborne viruses (Verreault et al., 2010).

Aerosols are impacted on PUF which is often considered superior to other substrates such as glass bead beds, cellulose filters and other solid substrates. In particular, the PUF has minimum particle bounce and re-entrainment losses and has high collection efficiency without the need for greasing or oiling the substrate (Crook et al., 1997; Kavouras and Koutrakis, 2001; Lee et al., 2005). Furthermore, there is no pressure drop increase with particle accumulation, and it is easy to recover the impacted particles from the PUF.

In contrast to the ASAP bioaerosol system described in this study, some samplers utilize a combination of immunoassay and PCR to increase reliability and minimize the possibility of false positives (Hindson et al., 2005). Furthermore, the number of agents that can be detected in a sample is limited by the number of primer/probe sets used for multiplexing. If the primer/probe for a specific pathogen is not included in the mix, it would not be detected. It is expected that in the future pathogen detection will be based primarily on next-generation sequencing (NGS; Frey et al., 2014; Lecuit and Eloit, 2014). A key advantage of NGS is that it allows identification of a range of organisms (bacteria, viruses, fungi). Subsequent adaptations to couple the ASAP bioaerosol sampler with NGS platforms can potentially further enhance its accuracy, sensitivity and spectrum of detection.

The ASAP sampler is highly portable (32 cm/side; 13.6 kg), quiet in operation and has low energy usage (battery operable). The ASAP system equipment is extremely user friendly and allows changing the default settings of the machine to add attachments such as a GPS, wireless and Ethernet communication, temperature, and wind sensor, video exposure monitoring with regular and infrared cameras to identify anyone with fever who may be a carrier of a pathogen, and real-time sensors for radioactivity. ASAP can be controlled using an external trigger. All of these aspects of the ASAP 2800 system would make it a dynamic and powerful tool in the detection of airborne pathogens. The operation of the sampler can be adjusted depending on the requirement of the facility, for example – 1 h for each PUF for a total 8 h run or half an hour per PUF for a 4 h run. Sample analysis can be done once every 4–8 h or more frequently depending on the need of the facility. Once released, the virus-loaded fine bioaerosol particles can remain suspended for hours (Yang et al., 2011) and available for detection by bioaerosol samplers. At the end of each run, the sealed iBASS cartridge can be aseptically removed and immediately analyzed for the presence of any alarming genetic material. The rotating iBASS cartridge can hold as many as eight different sampling PUFs along with a blank and negative control. The option of eight different samples allows the selection of specific times to acquire samples. Since there is no cross contamination between the PUFs in an iBASS cartridge, the sampling at different times can be useful to detect the time period when the airborne pathogen was brought into the area. Standard operating procedures for an ASAP system have already been developed by Lovelace Respiratory Research Institute.

For maximum efficacy in larger environments such as airports or stadiums, the bioaerosol samplers can be installed at strategic locations and work in conjunction with additional security surveillance such as video, personnel, and canine units. The ASAP bioaerosol sampler can also be used for evaluation of microbial contamination in the working environments in agricultural (Popescu et al., 2014; Sondergaard et al., 2014) or industry (Skora et al., 2014) settings, investigation of microbial metagenomics at distinct environments (Be et al., 2015) or in hospitals to track, identify, and control nosocomial infections (Sudharsanam et al., 2012).

Overall, these results suggest that the ASAP bioaerosol sampler is a promising system for monitoring ambient air that has been proven to work with viral pathogens. Additional testing to evaluate its feasibility in real world conditions such as small airports, hospitals, and military installations is recommended for its further development and subsequent use for the detection of real pathogens. This study also presents an ideal surrogate virus that can potentially be used in additional studies investigating viral aerosol sampling and detection. BAd-ΔE1E3 is a DNA virus that is quite stable in the environment. BAdV3 does not cause any harm to humans and to ensure additional safety it has been genetically modified to make it replication defective.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.