An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.
This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.
The purpose of this method is to provide a highly sensitive bioaerosol sampling platform to facilitate molecular detection of negatively-charged viruses from bioaerosols. Microorganisms, including viral particles, can survive in bioaerosols for extended periods of time1. Bioaerosols can travel over relatively long distances and maintain viability and infectivity, as evidenced by an outbreak of Legionnaires' disease that originated from industrial cooling towers located at a distance of 6 km from the affected individuals and resulted in 18 fatalities2. Indirect transmission of viruses to humans mediated by bioaerosols can occur in multiple settings and has been demonstrated for norovirus outbreaks in schools and restaurants3,4. Similarly, bioaerosol transmission of viruses can occur in agricultural settings such as in swine and poultry farms, with this transmission route being considered as a major factor in the movement of viruses between production facilities5,6,7,8,9.
Effective sampling of virus-laden bioaerosols allows for improvement in rapid diagnostics and preparedness for outbreak prevention, as shown in demonstrations in which H5 influenza A viruses were detected from bioaerosols in live animal markets in China and the United States10,11. Current bioaerosol sampling technologies involve a number of different particle capture principles, and can be broadly categorized into impingers, cyclones, impactors, and filters12. It is beyond the scope of this protocol to exhaustively cover all advantages and disadvantages of these platforms for sampling of viruses from bioaerosols; however, it can be stated that the majority of these sampling devices have not been optimized for the collection of viruses and bacteriophages13. Furthermore, infectivity of viral particles is often negatively affected, with liquid impingers considered to maintain viral infectivity more effectively than sampling devices such as solid impactors or filters14. However, one disadvantage of liquid impingement is the target dilution effect, which occurs because viruses are collected in relatively large volumes (typically ≥20 mL) of liquid in the collection vessel. Another important disadvantage involves the suboptimal efficiency of liquid impingers to concentrate particles <0.5 µM in size15. However, capture efficiency of these devices can be improved by immobilization on solid matrices, as immobilization can enhance preservation of viral nucleic acids and viral infectivity16,17.
We have previously demonstrated that anion exchange resin is an effective tool for the capture and concentration of viruses from liquid matrices, including F-RNA bacteriophages, hepatitis A virus, human adenovirus, and rotavirus18,19,20. As defined by the manufacturer, the anion exchange resin utilized in this work is a macroreticular polystyrene strong base anion exchange resin in which functionalized quaternary amine groups mediate attraction and capture of anions in a liquid medium21. Consequently, the anion exchange resin is expected to capture viruses with net-negative surface charges, including many enteric viruses, influenza viruses and other viruses relevant to public and animal health.
The current protocol involves the addition of anion exchange resin to a liquid impinger. In this system, the resin serves as a secondary concentration step for viral particles captured in the impinger liquid. Nucleic acids can then be directly eluted in small volumes, providing a concentrated sample for molecular analyses. Thus, the main advantage of this method is the improvement in viral detection sensitivity, primarily through reduction in sample volume. Additionally, due to the inherent non-specific capture of negatively-charged viruses, the method is likely applicable for detection of a large number of viruses of interest. Here, the method is demonstrated for vaccine strains of type A and type B influenza viruses and the FRNA coliphage MS2 (MS2). These viruses are subsequently detected using standard qRT-PCR assays as previously described22. The end-point user should not expect to encounter difficulties in performing this method, because modifications to currently existing equipment do not constitute major disruptions to the conventional flow of bioaerosol sampling and analysis.
1. Setup of the Bioaerosol Chamber (See Figure 2)
2. Model Bioaerosolization Experiments (see Figure 3)
3. Nucleic Acid Extraction and qRT-PCR Detection
Figure 1 demonstrates the principle behind charge-based capture of viruses from bioaerosols via inclusion of resin in liquid-based impingers. Figure 2 shows the setup of the custom-built bioaerosol chamber. Figure 3 describes the steps involved in setting up the aerosolization experiment and measures to ensure quality control. Figure 4 shows amplification curves for qRT-PCR detection of negatively-charged viruses captured from bioaerosols. Table 1 shows the primers and probes used for qRT-PCR for the example viruses used in this study.
Using MS2 and influenza viruses contained within the influenza vaccine as models in the custom bioaerosol chamber, the liquid impingers modified with anion exchange resin allowed for approximately 3x to 9x improvement in viral detection, depending on the specific virus, compared to direct testing of impingement liquid22. As shown in representative qRT-PCR amplification curves (Figure 4), detection of type A influenza viruses could be achieved up to 3.26 cycles sooner when anion exchange resin was used compared to direct testing of the impingement liquid. Considering that in a qRT-PCR that is 100% efficient, a gain of one Ct corresponds to a 2-fold increase in target concentration, this difference is equivalent to a 9.58x improvement in detection.
Figure 1: Liquid impingement coupled with anion exchange resin. Each of the modified bioaerosol sampling devices incorporates an anion exchange resin into its design. Each resin bead is a 0.5 to 1.0 mm sphere with a porous surface, a characteristic that increases the anion exchange surface area. Functional quaternary ammonium groups allow for capture of ions in liquid media, mediating capture of viruses with net-negative surface charges, including MS2 coliphages and influenza viruses, which are used in this protocol as model organisms (a tailed bacteriophage is drawn here as an example for captured viruses). Nucleic acid isolation is then performed directly from the resin (using commercially available kits), with the sample suitable for most molecular detection strategies (qRT-PCR employed here). Please click here to view a larger version of this figure.
Figure 2: Custom bioaerosol chamber schematic. A: 6-jet nebulizer supplied with filtered and dried air at a generating pressure of 6.9 kPa; B: Axial fans to facilitate uniform bioaaerosol mixing; C: Aerosol monitor to identify the relative bioaerosol concentrations; D: Liquid impinger with resin; E: Liquid impinger without resin; F: Thermal anemometer to measure wind speed; G: Air quality monitor to measure other environmental variables including temperature, carbon dioxide, and percent relative humidity; H: Active sampling pumps calibrated to 12.5 L/min; I: Pneumatically sealed glove ports for positioning sampling equipment within the bioaerosol chamber; J: Observation window. This figure was reused from previous work with permission from the publisher22. Please click here to view a larger version of this figure.
Figure 3: Workflow of the bioaerosolization experiment. Please click here to view a larger version of this figure.
Figure 4: Anion exchange resin improves qRT-PCR based detection of negatively-charged viruses in bioaerosols. In this example, type A influenza viruses at an inoculum concentration of 103.5 FFU/mL were aerosolized into the custom bioaerosol chamber until the bioaerosol mass concentration reached 5 mg/m3. Liquid impingers with and without resin were simultaneously engaged to each sample (500 L) of the bioaerosol. Viral RNA was obtained from the anion exchange resin or from the liquid of the impinger and assayed by qRT-PCR. The experiment was repeated in triplicate. Nucleic acids obtained from resin samples were detected at an average Ct of 26.76, whereas impinger liquid samples were detected at an average Ct of 30.02 (ΔCt of 3.26). This difference in Ct is equivalent to a 9.58x improvement in detection using the anion exchange resin. Please click here to view a larger version of this figure.
Viruses | Primer name | Primer sequence | Reference | |
MS2 | GI Fwd | 5'-ATCCATTTTGGTAACGCCG-3' | 23 | |
GI Probe | 5'-(6-FAM)-TAGGCATCTACGGGGACGA-(BBQ)-3' | |||
GI Rev | 5'-TGCAATCTCACTGGGACATAT-3' | |||
IAC Fwd (46F) | 5'-GACATCGATATGGGTGCCG-3' | |||
IAC Probe | 5'-(Cy5)-CACATTCACCAGGGAGACGCATGAGA-(BBQ)-3' | |||
IAC Rev (186R) | 5'-CGAGACGATGCAGCCATTC-3' | |||
type A influenza viruses | CDC universal influenza A virus forward primer | 5'-GAC CRA TCC TGT CAC CTC TGA C-3' | 24 | |
CDC universal influenza A virus reverse primer | 5'-AGG GCA TTY TGG ACA AAK CGT CTA-3' | |||
CDC universal influenza A virus probe | 5'-(FAM)-TGC AGT CCT CGC TCA CTG GGC ACG-(BHQ1)-3' | |||
type B influenza viruses | CDC universal influenza B virus forward primer | 5'-TCCTCAACTCACTCTTCGAGCG-3' | 24 | |
CDC universal influenza B virus reverse primer | 5'-CGGTGCTCTTGACCAAATTGG-3' | |||
CDC universal influenza B virus probe | 5'-(JOE)-CCAATTCGAGCAGCTGAAACTGCGGTG-(BHQ1)-3' |
Table 1: Primers and probes used in this study. All primers and probes were from Friedman et al. 201123 and Selvaraju & Selvarangan 201024.
This protocol outlines a method for sensitive viral capture from bioaerosols using modified liquid impingers. The method is optimized for detection and quantification of the viral load in bioaerosols. The specific modification demonstrated here involves the addition of anion exchange resin to liquid contained within a common liquid impinger. This method was developed for its simplicity in downstream sample processing, whereas other sample processing techniques such as centrifugation, filtration, and precipitation-based methods do not offer such an advantage. Extraction of nucleic acids is performed directly from the resin, obviating the need for high-volume elutions and multiple sample preparation steps, ultimately contributing to improved detection sensitivity of the method via reduction in the effective sample volume. Furthermore, it has been shown that immobilization of viral particles on solid surfaces can contribute to increased stability of viral nucleic acid or retention of viral infectivity17,25. Immobilization may further contribute to increased capture efficiency by reducing losses due to reaerosolization16. The method can be easily coupled with downstream molecular analyses (qRT-PCR demonstrated here). The anion exchange resin may be field-extracted and shipped to a reference laboratory for analysis, while air-sampling equipment can stay dedicated to monitoring in the sampling area. As the principle behind the procedure involves the non-specific capture of viruses carrying a net-negative surface charge, the method is expected to be amenable to the detection of multiple pathogenic viruses of importance to human health and veterinary medicine. As reviewed by Michen and Graule26, a large number of viruses important to public and animal health have net-negative surface charges, with few known exceptions, such as certain strains of rotaviruses and polioviruses.
This protocol does not directly evaluate the effect of environmental conditions on the sensitivity of detection; however, when air sampling is conducted in field settings, it is conceivable that variables such as relative humidity and temperature may affect collection efficiencies across multiple types of air samplers12. Thus, conditions within the bioaerosol chamber may be modified to suit these environmental parameters as needed. The anion exchange resin method has been shown to effectively concentrate multiple viruses from water samples with a wide range of physicochemical properties, indicating that interference from particulate matter, bacteria, and other environmental contaminants is expected to be minimal18. Additionally, collection buffers may require optimization for specific viruses, as in some cases it has been shown that optimizing the collection buffer can lead to improved retention of viral stability27. A main disadvantage of the method as presented is the fact that it cannot determine viral infectivity. Another disadvantage of this protocol is the inability to assess potentially negative effects of nebulization and air sampling on viral stability, including desiccation, shearing, and non-specific adsorption to bioaerosol chamber surfaces22.
The modified bioaerosol sampling devices could be amenable to field-based sampling in multiple settings, including hospitals, schools, agricultural operations, and other venues. Because modifications are made to existing air sampling equipment, it is expected the modified devices would be easily adoptable by operators currently sampling bioaerosols for viruses. The protocol is demonstrated here in a custom-built bioaerosol chamber using MS2 and influenza viruses, which are useful targets for method development due to the fact that they represent examples of non-enveloped and enveloped viruses, MS2 is recognized as a surrogate of enteric viruses and are commonly employed in evaluation of impingers22,26,27,28,29.
Future work will involve optimization of sampling conditions (i.e., air sampling velocity, collection buffers) and inclusion of additional relevant viruses in testing protocols.
The authors have nothing to disclose.
This work was supported by funding from the CDC/NIOSH High Plains Intermountain Center for Agricultural Health and Safety (5U54OH008085) and the Colorado Bioscience Discovery Evaluation Grant Program (14BGF-16).
Escherichia coli bacteriophage MS2 (ATCC 15597-B1) | American Type Culture Collection | ATCC 15597-B1 | |
FluMist Quadrivalent | AstraZeneca | Contact manufacturer | Viral constitutents of this vaccine are subject to change on an annual basis |
CFX96 Touch Real-Time PCR Detection System | Bio-Rad | 1855195 | |
Primers and probes | Integrated DNA Technologies | NA | |
0.2 µM sterile filter | NA | NA | |
1 L pyrex bottles or equivalent | NA | NA | |
1 mL pipet tips | NA | NA | |
1 mL pipettor | NA | NA | |
50 mL serological pipet | NA | NA | |
PCR tubes | NA | NA | |
Pipet-aid or equivalent | NA | NA | |
QIAamp Viral RNA Mini Kit | Qiagen | 52904 | |
QuantiTect Probe RT-PCR Kit | Qiagen | 204443 | |
Amberlite IRA-900 chloride form | Sigma-Aldrich | 216585-500G | |
Phosphate buffered saline | Sigma-Aldrich | P5368-10PAK | |
Water (molecular biology grade) | Sigma-Aldrich | W4502-1L | |
Eppendorf DNA LoBind Microcentrifuge Tubes | ThermoFisher | 13-698-791 | |
Falcon 50 mL Conical Centrifuge Tubes | ThermoFisher | 14-432-22 | |
Falcon Polypropylene Centrifuge Tubes | ThermoFisher | 05-538-62 | |
SuperScript III Platinum One-Step qRT-PCR Kit w/ROX | ThermoFisher | 11745100 | |
SKC Biosampler 20 mL, 3-piece glass set | SKC Inc. | 225-9593 | |
Vac-u-Go sample pumps | SKC Inc. | 228-9695 | |
Collison nebulizer (6-jet) | BGI Inc. | NA | |
HEPA capsule | PALL | 12144 | |
Q-TRAK indoor air quality monitor 8554 | TSI Inc. | NA | |
Alnor velometer thermal anemometer AVM440-A | TSI Inc. | NA | |
SidePak AM510 personal aerosol monitor | TSI Inc. | NA | |
Bioaerosol chamber | NA | NA |