Detection of Viruses from Bioaerosols Using Anion Exchange Resin
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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 m…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 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 |
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