Name: a small volume procedure for viral concentration from water
Uploaded: 2015-02-03T13:00:00
Description: Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.

1. Preparation of Glassware and Filter Housings
<ol style="margin-left: 40px;">
	<li>Unless otherwise noted, sterilize all glassware, filter housings and solutions at 121 &#176;C for 15 min. To ensure sterility, cover all openings or exposed surfaces with either aluminum foil or tape-secured paper prior to sterilization.</li>
	<li>Assemble filtration apparatus by attaching filter housing (47 mm diameter) to 1 L side arm Erlenmeyer flask. Collect filters required: 47 mm diameter electropositive/electronegative disc filt...

<ol>
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C., Fout, G. S. <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+the+celite+secondary+concentration+procedure+and+an+alternate+elution+buffer+for+the+recovery+of+enteric+adenoviruses+40+and+41.">Evaluation of the celite secondary concentration procedure and an alternate elution buffer for the recovery of enteric adenoviruses 40 and 41.</a> Journal of Virological Methods. 179, 423-428 (2012).</li><li>Katzenelson, E., Fattal, B., Hostovesky, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organic+flocculation:+an+efficient+second-step+concentration+method+for+the+detection+of+viruses+in+tap+water.">Organic flocculation: an efficient second-step concentration method for the detection of viruses in tap water.</a> Applied and Environmental Microbiology. 32, 638-639 (1976).</li><li>Fout, G. S., Schaefer, F. W., Messer, J. W., Dahling, 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=.">.</a> ICR microbial laboratory manual. 178, (1996).</li><li>Calgua, B., 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=Development+and+application+of+a+one-step+low+cost+procedure+to+concentrate+viruses+from+seawater+samples.">Development and application of a one-step low cost procedure to concentrate viruses from seawater samples.</a> Journal of Virological Methods. 153, 79-83 (2008).</li><li>Calgua, B., 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=Detection+and+quantification+of+classic+and+emerging+viruses+by+skimmed-milk+flocculation+and+PCR+in+river+water+from+two+geographical+areas.">Detection and quantification of classic and emerging viruses by skimmed-milk flocculation and PCR in river water from two geographical areas.</a> Water Research. 47, 2797-2810 (2013).</li><li>McMinn, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+adenovirus+40+and+41+recovery+from+tap+water+using+small+disk+filters.">Optimization of adenovirus 40 and 41 recovery from tap water using small disk filters.</a> Journal of Virological Methods. 193 (2), 284-290 (2013).</li><li>Brinkman, N. E., Haffler, T. D., Cashdollar, J. L., Rhodes, E. 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+methods+using+celite+to+concentrate+norovirus,+adenovirus+and+enterovirus+from+wastewater.">Evaluation of methods using celite to concentrate norovirus, adenovirus and enterovirus from wastewater.</a> Journal of Virological Methods. 193, 140-146 (2013).</li><li>Albinsson, B., Kidd, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+type+41+lacks+an+RGD+alpha(v)-integrin+binding+motif+on+the+penton+base+and+undergoes+delayed+uptake+in+A549+cells.">Adenovirus type 41 lacks an RGD alpha(v)-integrin binding motif on the penton base and undergoes delayed uptake in A549 cells.</a> Virus Research. 64, 125-136 (1999).</li><li>He, C., Lian, J. S., Jiang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electronic+structures+and+hydrogen+bond+network+of+high-density+and+very+high-density+amorphous+ices.">Electronic structures and hydrogen bond network of high-density and very high-density amorphous ices.</a> The Journal of Physical Chemistry. B. 109, 19893-19896 (2005).</li><li>Sedmak, G., Bina, D., Macdonald, J., Couillard, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nine-year+study+of+the+occurrence+of+culturable+viruses+in+source+water+for+two+drinking+water+treatment+plants+and+the+influent+and+effluent+of+a+Wastewater+Treatment+Plant+in+Milwaukee,+Wisconsin.">Nine-year study of the occurrence of culturable viruses in source water for two drinking water treatment plants and the influent and effluent of a Wastewater Treatment Plant in Milwaukee, Wisconsin.</a> 71, 1042-1050 (2005).</li><li>Soto-Beltran, M., Ikner, L. A., Bright, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effectiveness+of+poliovirus+concentration+and+recovery+from+treated+wastewater+by+two+electropositive+filter+methods.">Effectiveness of poliovirus concentration and recovery from treated wastewater by two electropositive filter methods.</a> Food and Environmental Virology. 5, 91-96 (2013).</li><li>Haramoto, E., Katayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+acidic+elution+to+virus+concentration+using+electropositive+filters.">Application of acidic elution to virus concentration using electropositive filters.</a> Food and Environmental Virology. 5, 77-80 (2013).</li><li>Lee, H., 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+electropositive+filtration+for+recovering+norovirus+in+water.">Evaluation of electropositive filtration for recovering norovirus in water.</a> Journal of Water and Health. 9, 27-36 (2011).</li><li>Gibbons, C. D., Rodriguez, R. A., Tallon, L., Sobsey, M. 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+positively+charged+alumina+nanofibre+cartridge+filters+for+the+primary+concentration+of+noroviruses,+adenoviruses+and+male-specific+coliphages+from+seawater.">Evaluation of positively charged alumina nanofibre cartridge filters for the primary concentration of noroviruses, adenoviruses and male-specific coliphages from seawater.</a> Journal of Applied Microbiology. 109, 635-641 (2010).</li><li>Hill, V. 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=Development+of+a+rapid+method+for+simultaneous+recovery+of+diverse+microbes+in+drinking+water+by+ultrafiltration+with+sodium+polyphosphate+and+surfactants.">Development of a rapid method for simultaneous recovery of diverse microbes in drinking water by ultrafiltration with sodium polyphosphate and surfactants.</a> Applied and Environmental Microbiology. 71, 6878-6884 (2005).</li><li>Polaczyk, A. L., Roberts, J. M., Hill, V. 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L., 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=Round+robin+investigation+of+methods+for+the+recovery+of+poliovirus+from+drinking+water.">Round robin investigation of methods for the recovery of poliovirus from drinking water.</a> Applied and Environmental Microbiology. 47, 144-150 (1984).</li><li>Schwab, K. J., De Leon, R., Sobsey, M. 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Electropositive filters are useful in concentrating viruses from water; however these filters can differ in their structure and composition which could in turn alter their effectiveness. Compounding this problem, capsid structures and charges vary between virus strains requiring concentration techniques be tailored to ensure optimal recovery 15. Through simple modifications of the existing concentration techniques (e.g., electropositive filters, beef extract elution), more effective concentration of t...

Human enteric viruses are important causative agents of waterborne diseases 1-3, but are generally present in low numbers in contaminated environmental waters, making their detection difficult without concentration. Procedures used to concentrate viruses typically include a filtration step, followed by filter elution, and secondary concentration of the filter eluate. A common filtration procedure relies on use of charged membranes such as electropositive filters (recently reviewed in 4,5). These filters rely on capturing viruses suspended in water using electrostatic interactions between the filter surface (positively charged) and targeted virus particles (negatively charged). Two electropositive filters that are commercially available rely on this technology, the glass/cellulose and nano-alumina/glass fiber filters. The glass/cellulose filter costs are up to 10 times that of the nano-alumina/glass fiber, which limit the use of the glass/cellulose filters for routine virus monitoring. Recent studies have concluded differences are nominal between these two filters in recovery of enteroviruses from ambient water 6,7, justifying the use of a cheaper filter alternative. Other filter options such as electronegative and glass-wool filters have been studied, however, they either require the pretreatment of source water (electronegative filters) or are not commercially available (glass-wool filters). The development of virus concentration procedures has mostly focused on optimizing primary concentration techniques (filters) in order to improve virus recoveries from water. However, secondary concentration procedures, which reduce the volume of eluant typically from 1 L to milliliter volumes, can also have a significant impact on virus recoveries 8.
Secondary concentration of enteric viruses typically relies on a flocculating agent such as some types of beef extract (organic flocculation) or skimmed milk flocculation 9-12 to remove virus particles from filter surfaces. Recently, another secondary concentration procedure using beef extract coupled with the addition of celite (fine particle) has shown promise for recovering adenovirus, enterovirus, and norovirus 8,13,14. Celite concentration works under similar principles to that of the organic flocculation method in that virus particles attach to and are released from particles (floc or celite) by altering the pH of the suspension solution. Comparisons between these two secondary concentration techniques have been evaluated in recovery of spiked adenovirus (AdV) types 40 and 41 8. This study concluded that the two secondary concentration techniques were statistically similar in recovery of adenoviruses. However, the organic flocculation method requires a 30 min. incubation at pH 3.5, while the celite technique requires a shorter incubation (10 min) at pH 4.0. The organic flocculation also requires the use of expensive laboratory equipment (centrifuges) to collect floc particles during tertiary concentration, the celite technique in contrast uses only basic laboratory equipment (vacuum filtration) to separate celite particles from suspension.
Certain combinations of filters and secondary elution techniques can also affect virus recoveries. One study concluded that certain combinations of primary (electropositive filters) and secondary concentration techniques (celite or organic flocculation) had a significant impact of recovery of adenovirus 13. These findings suggest that optimization is required in order to optimally recover target virus from a given water matrix when using these techniques. Optimization is a time consuming, arduous process many researchers actively avoid since numerous variables will be evaluated (filter type/brand, pH elution solution, celite/organic flocculation).
For this study, a procedure was developed to identify optimal conditions for virus concentration from water using spiked human adenovirus strains 40 and 41. Presumably, since each virus type displays a unique capsid morphology and specific capsid charge, concentration protocols may need to be optimized for every virus target in order to attain optimal viral recovery. This study provides an approach for AdV 40 and 41 concentration by: 1) evaluating virus recoveries in tap water using electropositive filter discs followed by 2) evaluation of an established organic flocculation method versus the celite technique as a secondary concentration, and 3) evaluation of elution buffers for tertiary concentration.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Name of Reagent/ Equipment</td>
			<td style="width:143px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:295px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adenovirus 40 stock</td>
			<td style="width:143px;">ATCC</td>
			<td style="width:119px;">VR-931</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Adenovirus 41 stock</td>
			<td style="width:143px;">ATCC</td>
			<td style="width:119px;">VR-930</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Thiosulfate</td>
			<td style="width:143px;">Fluka Chemical Co.</td>
			<td style="width:119px;">72051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Celites #577</td>
			<td style="width:143px;">Fluka Chemical Co.</td>
			<td style="width:119px;">22142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">NanoCeram 47mm</td>
			<td style="width:143px;">Argonide</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">1MDS 47mm</td>
			<td style="width:143px;">3M</td>
			<td style="width:119px;">6408502</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">AP-20 Prefilter 47mm</td>
			<td style="width:143px;">Millipore Corp.</td>
			<td style="width:119px;">AP2004700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Glycine&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">50046-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Sodium Polyphosphate</td>
			<td style="width:143px;">Acros Organics</td>
			<td style="width:119px;">390930010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Trypsin</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:119px;">25200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">PBS</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Hydrochloric Acid</td>
			<td style="width:143px;">Fisher</td>
			<td style="width:119px;">A481-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">BBL Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:119px;">212303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Difco Beef Extract</td>
			<td style="width:143px;">BD Biosciences</td>
			<td style="width:119px;">211520</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:231px;">ABI 7900 Real-time PCR system</td>
			<td style="width:143px;">ABI</td>
			<td style="width:119px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Stainless Steel Filter Housing</td>
			<td style="width:143px;">Millipore Corp.</td>
			<td>XX2004720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">Blood DNA Extraction Kit</td>
			<td style="width:143px;">Qiagen&#160;</td>
			<td style="width:119px;">51104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">EPA MPN Calculator</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.epa.gov/nerlcwww/online.html</td>
		</tr>
	</tbody>
</table>

a small volume procedure for viral concentration from water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

Celite selection
Three different types of celite were tested prior to the selection of the best performing variant. Celites with fine to medium sized particles produced the highest adenovirus recoveries. Use of larger celites resulted in lower recoveries for both AdV40 and 41 (range 32%-100%) (Figure 1). Average recoveries of adenovirus 40 were 144% &#177; 52% (fine), 115% &#177; 28% (medium) and 82% &#177; 53% (large) particle celites and for AdV41, 132% &#177; 39% (fine), 83...

Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

A Small Volume Procedure for Viral Concentration from Water

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

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