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).

1. Setup of the Bioaerosol Chamber (See Figure 2)
<ol>
	<li>Pre-load the liquid impingers with 20 mL of 0.01 M phosphate buffered saline, pH 7.5 (PBS).
	<ol>
		<li>Add 0.5 g of anion exchange resin and suspend within the PBS of one of the liquid impingers, with another liquid impinger serving as a control.</li>
	</ol>
	</li>
	<li>Position liquid impingers in parallel inside the bioaerosol chamber using clamp stands with aerosol inlets facing the nebulizer. 
	NOTE: See...

<ol>
	<li>Pirtle, E. C., Beran, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+survival+in+the+environment.">Virus survival in the environment.</a> Revue scientifique et technique (International Office of Epizootics). 10 (3), 733-748 (1991).</li><li>Nguyen, T. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+community-wide+outbreak+of+legionnaires+disease+linked+to+industrial+cooling+towers--how+far+can+contaminated+aerosols+spread?.">A community-wide outbreak of legionnaires disease linked to industrial cooling towers--how far can contaminated aerosols spread?.</a> The Journal of Infectious Diseases. 193 (1), 102-111 (2006).</li><li>Marks, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+airborne+transmission+of+Norwalk-like+virus+(NLV)+in+a+hotel+restaurant.">Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant.</a> Epidemiology and Infection. 124 (3), 481-487 (2000).</li><li>Marks, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+school+outbreak+of+Norwalk-like+virus:+Evidence+for+airborne+transmission.">A school outbreak of Norwalk-like virus: Evidence for airborne transmission.</a> Epidemiology and Infection. 131 (1), 727-736 (2003).</li><li>Corzo, C. A., Culhane, M., Dee, S., Morrison, R. B., Torremorell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Airborne+detection+and+quantification+of+swine+influenza+a+virus+in+air+samples+collected+inside,+outside+and+downwind+from+swine+barns.">Airborne detection and quantification of swine influenza a virus in air samples collected inside, outside and downwind from swine barns.</a> PLoS One. 8 (8), e71444 (2013).</li><li>Anderson, B. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioaerosol+sampling+in+modern+agriculture:+A+novel+approach+for+emerging+pathogen+surveillance.">Bioaerosol sampling in modern agriculture: A novel approach for emerging pathogen surveillance.</a> The Journal of Infectious Diseases. 214 (4), 537-545 (2016).</li><li>Hietala, S. K., Hullinger, P. J., Crossley, B. M., Kinde, H., Ardans, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+air+sampling+to+detect+exotic+Newcastle+disease+virus+in+two+California+commercial+poultry+flocks.">Environmental air sampling to detect exotic Newcastle disease virus in two California commercial poultry flocks.</a> Journal of Veterinary Diagnostic Investigation. 17 (2), 198-200 (2005).</li><li>Jonges, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wind-mediated+spread+of+low-pathogenic+avian+influenza+virus+into+the+environment+during+outbreaks+at+commercial+poultry+farms.">Wind-mediated spread of low-pathogenic avian influenza virus into the environment during outbreaks at commercial poultry farms.</a> PLoS One. 10 (5), e0125401 (2015).</li><li>Otake, S., Dee, S. A., Jacobson, L., Torremorell, M., Pijoan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+aerosol+transmission+of+porcine+reproductive+and+respiratory+syndrome+virus+under+controlled+field+conditions.">Evaluation of aerosol transmission of porcine reproductive and respiratory syndrome virus under controlled field conditions.</a> The Veterinary Record. 150 (26), 804-808 (2002).</li><li>Wu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aerosolized+avian+influenza+A+(H5N6)+virus+isolated+from+a+live+poultry+market,+China.">Aerosolized avian influenza A (H5N6) virus isolated from a live poultry market, China.</a> The Journal of Infection. 74 (1), 89-91 (2017).</li><li>Choi, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+animal+markets+in+Minnesota:+A+potential+source+for+emergence+of+novel+influenza+A+viruses+and+interspecies+transmission.">Live animal markets in Minnesota: A potential source for emergence of novel influenza A viruses and interspecies transmission.</a> Clinical Infectious Diseases. 61 (9), 1355-1362 (2015).</li><li>Haig, C. W., Mackay, W. G., Walker, J. T., Williams, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioaerosol+sampling:+Sampling+mechanisms,+bioefficiency+and+field+studies.">Bioaerosol sampling: Sampling mechanisms, bioefficiency and field studies.</a> The Journal of Hospical Infection. 93 (3), 242-255 (2016).</li><li>Anderson, B. D., Lednicky, J. A., Torremorell, M., Gray, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+bioaerosol+aampling+for+airborne+virus+surveillance+in+swine+production+facilities:+A+mini+review.">The use of bioaerosol aampling for airborne virus surveillance in swine production facilities: A mini review.</a> Frontiers in Veterinary Science. 4, 121 (2017).</li><li>Verreault, D., Moineau, S., Duchaine, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+sampling+of+airborne+viruses.">Methods for sampling of airborne viruses.</a> Microbiology and Molecular Biology Reviews. 72 (3), 413-444 (2008).</li><li>Hogan, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+methodologies+and+dosage+assessment+techniques+for+submicrometre+and+ultrafine+virus+aerosol+particles.">Sampling methodologies and dosage assessment techniques for submicrometre and ultrafine virus aerosol particles.</a> Journal of Applied Microbiology. 99 (6), 1422-1434 (2005).</li><li>Yu, K. -. P., Chen, Y. -. P., Gong, J. -. Y., Chen, Y. -. C., Cheng, C. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+the+collection+efficiency+of+the+liquid+impinger+for+ultrafine+particles+and+viral+aerosols+by+applying+granular+bed+filtration.">Improving the collection efficiency of the liquid impinger for ultrafine particles and viral aerosols by applying granular bed filtration.</a> Journal of Aerosol Science. 101, 133-143 (2016).</li><li>Perez-Mendez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+a+simple+and+cost+effective+filter+paper-based+shipping+and+storage+medium+for+environmental+sampling+of+F-RNA+coliphages.">Evaluation of a simple and cost effective filter paper-based shipping and storage medium for environmental sampling of F-RNA coliphages.</a> J Virol Methods. 194 (1-2), 60-66 (2013).</li><li>Chandler, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field-based+evaluation+of+a+male-specific+(F+)+RNA+coliphage+concentration+method.">Field-based evaluation of a male-specific (F+) RNA coliphage concentration method.</a> Journal of Virological Methods. 239, 9-16 (2017).</li><li>Perez-Mendez, A., Chandler, J. C., Bisha, B., Goodridge, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+of+enteric+viruses+from+tap+water+using+an+anion+exchange+resin-based+method.">Concentration of enteric viruses from tap water using an anion exchange resin-based method.</a> Journal of Virological Methods. 206, 95-98 (2014).</li><li>Perez-Mendez, A., Chandler, J. C., Bisha, B., Goodridge, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+anion+exchange+resin-based+method+for+concentration+of+F-RNA+coliphages+(enteric+virus+indicators)+from+water+samples.">Evaluation of an anion exchange resin-based method for concentration of F-RNA coliphages (enteric virus indicators) from water samples.</a> Journal of Virological Methods. 204, 109-115 (2014).</li><li>Kammerer, J., Carle, R., Kammerer, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adsorption+and+ion+exchange:+Basic+principles+and+their+application+in+food+processing.">Adsorption and ion exchange: Basic principles and their application in food processing.</a> Journal of Agricultural and Food Chemistry. 59 (1), 22-42 (2011).</li><li>Chandler, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+the+improved+detection+of+aerosolized+influenza+viruses+and+the+male-specific+(F+)+RNA+coliphage+MS2.">A method for the improved detection of aerosolized influenza viruses and the male-specific (F+) RNA coliphage MS2.</a> Journal of Virological Methods. 246, 38-41 (2017).</li><li>Friedman, S. D., Cooper, E. M., Calci, K. R., Genthner, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+assessment+of+a+real+time+reverse+transcription-PCR+method+to+genotype+single-stranded+RNA+male-specific+coliphages+(Family+Leviviridae).">Design and assessment of a real time reverse transcription-PCR method to genotype single-stranded RNA male-specific coliphages (Family Leviviridae).</a> Journal of Virological Methods. 173 (2), 196-202 (2011).</li><li>Selvaraju, S. B., Selvarangan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+three+influenza+A+and+B+real-time+reverse+transcription-PCR+assays+and+a+new+2009+H1N1+assay+for+detection+of+influenza+viruses.">Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses.</a> Journal of Clinical Microbiology. 48 (11), 3870-3875 (2010).</li><li>Cademartiri, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immobilization+of+bacteriophages+on+modified+silica+particles.">Immobilization of bacteriophages on modified silica particles.</a> Biomaterials. 31 (7), 1904-1910 (2010).</li><li>Michen, B., Graule, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoelectric+points+of+viruses.">Isoelectric points of viruses.</a> Journal of Appled Microbiology. 109 (2), 388-397 (2010).</li><li>Turgeon, N., Toulouse, M. J., Martel, B., Moineau, S., Duchaine, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+bacteriophages+as+models+for+viral+aerosol+studies.">Comparison of five bacteriophages as models for viral aerosol studies.</a> Applied and Environmental Microbiology. 80 (14), 4242-4250 (2014).</li><li>Vergara, G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+FRNA+coliphages+as+indicators+of+human+enteric+viruses+in+a+tropical+urban+freshwater+catchment.">Evaluation of FRNA coliphages as indicators of human enteric viruses in a tropical urban freshwater catchment.</a> Water Research. 79, 39-47 (2015).</li><li>Tung-Thompson, G., Libera, D. A., Koch, K. L., de Los Reyes, F. L., Jaykus, L. 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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...

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&#39; 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 &#8805;20 mL) of liquid in the collection vessel. Another important disadvantage involves the suboptimal efficiency of liquid impingers to concentrate particles &#60;0.5 &#181;M 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.

<table>
	<tbody>
		<tr>
			<td>Escherichia coli bacteriophage MS2 (ATCC 15597-B1)</td>
			<td>American Type Culture Collection</td>
			<td>ATCC 15597-B1</td>
			<td></td>
		</tr>
		<tr>
			<td>FluMist Quadrivalent</td>
			<td>AstraZeneca</td>
			<td>Contact manufacturer</td>
			<td>Viral constitutents of this vaccine are subject to change on an annual basis</td>
		</tr>
		<tr>
			<td>CFX96 Touch Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>1855195</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers and probes</td>
			<td>Integrated DNA Technologies</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;M sterile filter</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 L pyrex bottles or equivalent</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL pipet tips</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL pipettor</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL serological pipet</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet-aid or equivalent</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp Viral RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>52904</td>
			<td></td>
		</tr>
		<tr>
			<td>QuantiTect Probe RT-PCR Kit</td>
			<td>Qiagen</td>
			<td>204443</td>
			<td></td>
		</tr>
		<tr>
			<td>Amberlite IRA-900 chloride form</td>
			<td>Sigma-Aldrich</td>
			<td>216585-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P5368-10PAK</td>
			<td></td>
		</tr>
		<tr>
			<td>Water (molecular biology grade)</td>
			<td>Sigma-Aldrich</td>
			<td>W4502-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf DNA LoBind Microcentrifuge Tubes</td>
			<td>ThermoFisher</td>
			<td>13-698-791</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 mL Conical Centrifuge Tubes&#160;</td>
			<td>ThermoFisher</td>
			<td>14-432-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Polypropylene Centrifuge Tubes</td>
			<td>ThermoFisher</td>
			<td>05-538-62</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III Platinum One-Step qRT-PCR Kit w/ROX</td>
			<td>ThermoFisher</td>
			<td>11745100</td>
			<td></td>
		</tr>
		<tr>
			<td>SKC Biosampler 20 mL, 3-piece glass set</td>
			<td>SKC Inc.</td>
			<td>225-9593</td>
			<td></td>
		</tr>
		<tr>
			<td>Vac-u-Go sample pumps</td>
			<td>SKC Inc.</td>
			<td>228-9695</td>
			<td></td>
		</tr>
		<tr>
			<td>Collison nebulizer (6-jet)</td>
			<td>BGI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPA capsule</td>
			<td>PALL</td>
			<td>12144</td>
			<td></td>
		</tr>
		<tr>
			<td>Q-TRAK indoor air quality monitor 8554</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Alnor velometer thermal anemometer AVM440-A</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>SidePak AM510 personal aerosol monitor</td>
			<td>TSI Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioaerosol chamber</td>
			<td>NA</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of viruses from bioaerosols using anion exchange resin

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.

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-...

Watch this Scientific Journal Video about Detection of Viruses from Bioaerosols Using Anion Exchange Resin at JoVE.com

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

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 ...

This method can help answer key questions about the type, level, and distribution of viruses in bioaerosols that are important for public and veterinary health. The main advantages of this method are its improvement on the sensitivity of molecular detection of viruses from bioaerosols, its field amenability, and its ease of use. Create suspensions of the experimental concentrations of viral particles of interest in 100 milliliter volumes of PBS within the glass vessels of a six-jet collision nebulizer.To set up the bioaerosol chamber, preload two liquid impingers with 20 milliliters of 0.01 molar PBS followed by the addition of 0.5 grams of anion exchange resin to a single impinger. Assemble the impinger. Install the suspensions within the chamber.Use clamp stands to position liquid impingers in parallel inside the bioaerosol chamber with the aerosol sampler inlets facing the nebulizer. Place a direct read instrument near the liquid impingers to monitor the environmental variables and place an additional direct read aerosol monitor near the liquid impingers to measure the mass concentration of the bioaerosol. Then, run the axial fans in the corners of the bioaerosol chamber to facilitate mixing of the aerosol.When the chamber is ready, start the instruments for measuring the variables within the bioaerosol chamber. Use a primary flow standard to calibrate the system to maintain the consistency between the sampling volumes. Seal the chamber for a 10 minute purge with HEPA filtered air.Deliver filtered, dried air to the nebulizer at 6.9 kilopascals of pressure to generate a target concentration of viral bioaerosols. When the target mass concentration of the aerosol has been reached, stop the nebulization. Then, use a HEPA filtered line located near the nebulizer to supply diluted air, taking care to maintain a constant pressure within the chamber and actively sample the bioaerosol chamber atmosphere for 40 minutes to achieve a sampling volume of 500 liters per sample.Calibration of the instrument is critical for determining the concentration of air contaminants. The sampling train should be calibrated before and after each sampling event. Pre and post calibrations should be within 5%of one another.Turn off the pumps at the end of the sampling. To isolate nucleic acids directly from the anion exchange resin, transfer all of the anion exchange resin containing liquid from the liquid impingers into individual, sterile 50 milliliter conical tubes. After allowing the resin to settle, slowly decant the liquid sample from the anion exchange resin, using a one milliliter pipette tip to remove any remaining liquid from the resin.Next, add 560 microliters of viral lysis buffer containing carrier RNA from a viral RNA isolation kit directly to the anion exchange resin, for a 10 minute incubation at room temperature with periodic mixing. At the end of the incubation, use a one milliliter pipette tip to transfer the viral lysate to a sterile 1.5 milliliter conical tube and proceed with the RNA isolation according to the manufacturer's instructions. Then, perform quantitative real-time PCR for detection of MS2 bacteriaphage and influenza A and B viruses using five microliters of nucleic acid eluent per sample.As shown in these representative qRT-PCR amplification curves, the detection of Type A influenza viruses can be achieved up to 3.26 cycles sooner when anion exchange resin is used, compared to direct testing of the impingement liquid. In a qRT-PCR that is 100%efficient, a gain of one threshold cycle corresponds to a two-fold increase in target concentration, thus the representative results indicate a 9.58 fold improvement in detection sensitivity. Once mastered, this technique can be completed in less than three hours if performed properly.While attempting this procedure, it's important to remember that when air sampling is conducted in the field, parameters such as relative humidity and temperature may affect collection efficiencies and that collection buffers may have to be optimized for different viruses. Following this procedure, other culture based methods can be utilized to answer additional questions about viral infectivity. After its development, this technique has paved the way for researchers and other professionals in fields of agricultural and public health, to explore alternative methods for viral capture and concentration from bioaerosols for risk assessment in viral disease surveillance.After watching this video, you should have a good understanding of how to capture and concentrate viruses from bioaerosols using modified liquid impingers, extract nucleic acids directly from the anion exchange resin and detect viruses using qRT-PCR. Don't forget that working with viral pathogens can be extremely hazardous and precautions such as using personal protective equipment and bio-safety level two or higher should always be taken when performing this procedure.

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JoVE Journal

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8.1K 조회수.  Colorado State University. This method can help answer key questions about the type, level, and distribution of viruses in bioaerosols that are important for public and veterinary health. The main advantages of this method are its improvement on the sensitivity of molecular detection of viruses from bioaerosols, its field amenability, and its ease of use. Create suspensions of the experimental concentrations of viral particles of interest in 100 milliliter volumes of PBS within the glass vessels of a six-jet collision ...