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 filters.</li>
	<li>Prepare 1 L of 1.5% beef extract (flocculating; produces floc particles when pH of solution is lowered to 3.5, or non-flocculating; which does not aggregate at pH 3.5), with 0.05 M glycine, by dissolving 15 g of beef extract in 1 L of deionized water and adding 3.75 g glycine. 
	NOTE: Non-flocculating beef extracts will require the addition of celite prior to tertiary concentration.</li>
	<li>Add elution additive sodium polyphosphate to beef extract at a 0.1% concentration.</li>
	<li>Add 1 M hydrochloric acid solution, drop wise, to mixing beef extract, pH solution to desired pH. Autoclave beef extract for 15-30 min at 121 &#176;C.</li>
	<li>Measure 0.1 g of fine, medium, or large particle celite (for use with non-flocculating beef extract only).</li>
	<li>Prepare vacuum/suction by attaching compatible tubing to Erlenmeyer side-arm flask and to the vacuum outlet.</li>
</ol>
2. Preparation of Solutions and Virus Stock
<ol style="margin-left: 40px;">
	<li>Prepare 1 L of 1 M HCl.</li>
	<li>Prepare 1 L of 1 M NaOH.</li>
	<li>Prepare 1x PBS solution (137 mM NaCl, 2.7 mM KCl, 1.47 mM K2PO4, and 4.3 mM NaH2PO4).
	<ol>
		<li>Adjust pH of 1x PBS to 9.0 using 1 M NaOH.</li>
	</ol>
	</li>
	<li>Prepare and dilute stock virus to 104-105 most probable number (MPN) ml-1 by mixing 1 ml of stock virus with 9 ml of PBS (pH 7), as previously described 8,13. Store at -80 &#176;C in 1 ml aliquots.
	<ol>
		<li>Sterilize virus stock by syringe filtering (0.22 &#956;m pore size) to remove potential contaminants.</li>
	</ol>
	</li>
	<li>Dechlorinated Tap Water
	<ol>
		<li>Measure 1 L of sterile tap water using a sterile graduated cylinder and pour into a 2 L beaker containing magnetic stir bar.</li>
		<li>Add 0.7 g of sodium thiosulfate and mix until granules dissolve. Adjust pH of tap water to 7-7.5 if needed using 1 M HCl or 1 M NaOH solution.</li>
		<li>Thaw virus spike (1 ml) and add entire volume of spike to 1 L of dechlorinated sterile tap water. Mix for 10 min.</li>
		<li>Prepare a method blank (dechlorinated tap water without adenovirus) with each experiment and process in the same manner as the spiked sample.</li>
	</ol>
	</li>
</ol>
3. Preparation of Millipore Filter Apparatus
<ol style="margin-left: 40px;">
	<li>Remove sterile covering over filter housing. Ensure proper fit between filter housing upper bowl and lower filter screen to avoid sample leakage during filtration.</li>
	<li>Remove sterile covering from 1 L Erlenmeyer flask. Ensure that the cork of the filter unit fits snugly in top opening of Erlenmeyer flask, creating a good seal.</li>
	<li>Remove top bowl of filter unit from lower filter screen, and place 47 mm electropositive filter disk squarely over screen with sterile forceps. Attach filter housing bowl to bottom screen and lock into place by twisting counterclockwise.</li>
</ol>
4. Tap Water Filtration (Primary Concentration)
<ol style="margin-left: 40px;">
	<li>Measure 100 ml of virus spiked tap water using sterile graduated cylinder.</li>
	<li>Pour 100 ml of virus spiked tap water into filter housing containing either a 47 mm pleated glass/cellulose filter or a nano-alumina/glass fiber disk filter.</li>
	<li>Allow water to slowly pass through filter. Apply gentle vacuum to remove remaining residuals of tap water from filter surface.</li>
	<li>If using trypsin treatment, add 5 ml of 0.25% trypsin to filter surface and incubate for 5 min.</li>
	<li>Wash trypsin from filter surface using 50 ml of sterile deionized water.</li>
</ol>
5. Virus Elution from Filter
<ol style="margin-left: 40px;">
	<li>Remove filter housing from 2 L Erlenmeyer flask, and place onto 250 ml side-arm flask.</li>
	<li>Measure 100 ml of beef extract using graduated cylinder. Slowly pour 100 ml of beef extract into filter housing.</li>
	<li>Allow beef extract to pour through filter, capturing beef extract in a small (250 ml) Erlenmeyer flask. Apply vacuum to pull through residual beef extract from filter.</li>
</ol>
6. Celite Secondary Concentration
<ol style="margin-left: 40px;">
	<li>Transfer flasks containing the eluted viruses to a stir plate. Add sterile magnetic stir bar and place on stir plate, mixing to create slight vortex.</li>
	<li>Add 0.1 g of celite powder to 100 ml of beef extract and allow celite to disperse. Place sterile pH probe into beef extract.</li>
	<li>Add drop wise 1 M HCl to beef extract to achieve pH 4.0. Allow to slowly mix for 10 min.</li>
	<li>Place 47 mm pre-filter onto filter housing (as previously described) using a 2 L Erlenmeyer flask.</li>
	<li>Pour 100 ml beef extract/celite mixture over pre-filter. Allow beef extract to pour through. Apply slight vacuum to pull through residual beef extract from filter.</li>
	<li>Place metal tube clip on end of filter housing spout. Attach sterile 15 ml polypropylene collection tube to metal tube clip. Place filter housing back into Erlenmeyer flask.</li>
	<li>Add 5 ml of 1x PBS buffer, pH 9.0 over celite collected on pre-filter.</li>
	<li>Allow PBS to drip through pre-filter. Apply slight vacuum to pull through residual PBS into 15 ml collection tube.</li>
</ol>
7. Organic Flocculation Secondary Concentration
<ol style="margin-left: 40px;">
	<li>Transfer the beakers containing the eluted virus to a stir plate. Add sterile magnetic stir bar to 100 ml of beef extract and mix to create slight vortex.</li>
	<li>Place sterile pH probe into beef extract.</li>
	<li>Add 1 M HCl drop wise to beef extract and adjust pH to 3.5. Mix for 30 min.</li>
	<li>Pour the eluate into 250 ml centrifuge conical tubes and centrifuge at 2,500 x g for 15 min at 4 &#186;C.</li>
	<li>Pour off supernatant taking care not to not to disrupt the pellet. Resuspend the pellet in 5 ml of 1x PBS, pH 9.</li>
	<li>Centrifuge the suspension at 4,000 x g for 10 min at 4 &#186;C. Pour off the supernatant and discard the pellet.</li>
	<li>Adjust the pH to 7-7.5, filter sterilize through 0.22 &#181;m syringe filter and freeze at -80 &#176;C.</li>
</ol>
8. Viral Nucleic Acid Extraction
<ol style="margin-left: 40px;">
	<li>Extract viral nucleic acids using an appropriate commercial kit according to manufacturer&#8217;s instructions.</li>
</ol>
9. AdV 40/41 qPCR
<ol style="margin-left: 40px;">
	<li>Use primers and probes, as well as reaction mix and thermal cycling profiles as published 8,13. 
	NOTE: Estimated limit of detection for the assay is approximately 5 qPCR units per reaction.</li>
	<li>Add components of PCR amplification mixtures at concentrations as follows: 10 mM Tris (pH 8.3), 50 mM KCl, 4.5 mM MgCl2, 10 mM dNTPs, 10 &#181;M primers and 1 &#181;M probe and 0.5 &#181;l of polymerase so that final volumes of 45 &#181;l can be added to appropriate number of wells within a 96-well PCR reaction plate.</li>
	<li>Load 45 &#181;l of PCR amplification mixture to each appropriate well of a 96-well, fast reaction PCR plate.</li>
	<li>Add 5 &#181;l of DNA extracted sample to appropriate wells of the PCR plate.</li>
	<li>Cover plate with heat resistant cover seal, spin the plate to move any droplets to bottom of PCR plate wells.</li>
	<li>Set PCR amplification cycles as follows: 95 &#176;C for 10 min, followed by 40 cycles of 95 &#176;C for 15 sec and 60 &#176;C for 1 min.</li>
</ol>
10. Data Analyses
<ol style="margin-left: 40px;">
	<li>Analyze triplicate measurements of the serial 5-fold dilutions of the samples spanning 1:5 to 1:625 dilution range in order to determine the most probable number (MPN) of the viral particles, as previously described 8,13.</li>
	<li>Analyze serial 5-fold dilutions of adenovirus experimental spikes spanning 1:5 to 1:15,625 dilution range in order to determine the most probable number (MPN) of the viral particles.</li>
	<li>Using the EPA MPN calculator (http://www.epa.gov/nerlcwww/online.html), adjust the calculator to account for type of dilution (1:5 or 1:10), number of dilutions performed, and number of replicate samples in dilution series.</li>
	<li>Perform log10 transformation of the data, followed by normalizing to a unit of volume (e.g., 1 ml, 100 ml, etc.). Calculate recoveries for given experiment by dividing number of viral particles present in sample concentrates with number of viral particles present in experimental spikes.</li>
	<li>Evaluate the effect of the different experimental variables (e.g., different celite types, pH ranges, different filter types and different secondary concentration techniques) on AdV recoveries by performing parametric or non-parametric statistical tests (e.g., paired t-test, one or two-way ANOVA) depending on the distribution of the data and the experimental design.</li>
</ol>

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No competing financial interests. 
The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

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><tbody><tr><td>Adenovirus 40 stock</td><td>ATCC</td><td>VR-931</td><td></td></tr><tr><td>Adenovirus 41 stock</td><td>ATCC</td><td>VR-930</td><td></td></tr><tr><td>Sodium Thiosulfate</td><td>Fluka Chemical Co.</td><td>72051</td><td></td></tr><tr><td>Celites #577</td><td>Fluka Chemical Co.</td><td>22142</td><td></td></tr><tr><td>NanoCeram 47 mm</td><td>Argonide</td><td>N/A</td><td></td></tr><tr><td>1MDS 47 mm</td><td>3M</td><td>6408502</td><td></td></tr><tr><td>AP-20 Prefilter 47 mm</td><td>Millipore Corp.</td><td>AP2004700</td><td></td></tr><tr><td>Glycine&amp;nbsp;</td><td>Sigma</td><td>50046-1KG</td><td></td></tr><tr><td>Sodium Polyphosphate</td><td>Acros Organics</td><td>390930010</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>P5368</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher</td><td>A481-212</td><td></td></tr><tr><td>BBL Beef Extract</td><td>BD Biosciences</td><td>212303</td><td></td></tr><tr><td>Difco Beef Extract</td><td>BD Biosciences</td><td>211520</td><td></td></tr><tr><td>ABI 7900 Real-time PCR system</td><td>ABI</td><td>N/A</td><td></td></tr><tr><td>Stainless Steel Filter Housing</td><td>Millipore Corp.</td><td>XX2004720</td><td></td></tr><tr><td>Blood DNA Extraction Kit</td><td>Qiagen&amp;nbsp;</td><td>51104</td><td></td></tr><tr><td>EPA MPN Calculator</td><td></td><td></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...

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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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Research

JoVE Journal

Environment

10.0K Views.  U.S. EPA. 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. 

Video: A Small Volume Procedure for Viral Concentration from Water

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

Immunology and Infection

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium1, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses2. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.

Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples&#160;is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 &#181;m and 0.2 &#181;m membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA.&#160;On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples&#160;due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types.

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Biology

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...

A Small Volume Procedure for Viral Concentration from Water

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