The publication of this article was supported by a grant from the Office of Research at the University of Tennessee. The authors thank the previous generation of students and researchers that have worked to refine these procedures. Research was supported by grants from the National Science Foundation (NSF-0851113, NSF-0825405 and NSF-0550485).

1. Ultrafiltration of Seawater to Generate "Virus-free" Water (Wilhelm &amp; Poorvin 2001)
<ol>
 <li>Approximately 20L of seawater / lakewater is collected as aseptically as possible.</li>
 <li>Water is serially prefiltered through 142-mm diameter polycarbonate 0.8 &mu;m filters that can be kept at -20 &deg;C for community analysis. Larger pore size filters may be used before this step for very productive systems.</li>
 <li>To obtain ultrafiltered water from an Amicon M12 system (Millipore) a 30 kDa-cutoff spiral cartridge is used to exclude all viruses, even small RNA viruses.</li>
 <li>The samples are processed and concentrated at ~ 25% speed with ~15-16 kPa of backpressure.</li>
 <li>The remaining sample of ~500 mL of water will contain a concentrated virus community (which can be saved for other experiments) while the remaining 19.5 L of virus-free water will be used for viral production assays.</li>
 <li>After each day of use, the Amicon M12 system must be cleaned to prevent damage to the membrane of the filter cartridge.</li>
 <li>If you are working with seawater, rinse the membrane out with at least 6L of Milli-Q water followed by a washing with 0.1N NaOH solution for 30-45 minutes.</li>
 <li>Again rinse the cartridge with at least 6L of Milli-Q water.</li>
 <li>When finished using the M12 system, the spiral cartridge should be stored in a 0.05M H3PO4 solution at 4&deg;C.</li>
</ol>
2. Virus Reduction Method for Viral Production (Wilhelm et al. 2002)
<ol>
 <li>Up to 500 mL of the seawater / lakewater sample with both host and viruses is obtained and placed in a sterifilter unit with a 0.2-&mu;m nominal pore-size low protein-binding filter (e.g., Durapore) placed on it.</li>
 <li>The sample is gently vacuum pressured at &lt;200 mmHg while continually resuspending the sample using a sterile transfer pipette to inhibit bacterial cells from concentrating on the filter.</li>
 <li>Slowly three volumes of ultrafiltrate are added to the bacterial suspension to significantly reduce the number of free viruses in the sample.</li>
 <li>The bacterial fraction is diluted back to 500 mL with virus-free water and divided into three replicates of 150 mL each and are placed in clear 250ml polycarbonate bottles.</li>
</ol>
3. Tangential Flow Filtration (TFF) Method for Viral Production (Weinbauer et al. 2002, Winget et al. 2005)
TFF represents an alternative approach to the viral reduction method.
<ol>
 <li>Approximately 500 mL of natural sample is collected as described above.</li>
 <li>This sample is concentrated using a 0.2-&mu;m nominal pore-size tangential flow filtration system.</li>
 <li>When the bacterial fraction is reduced to approximately 10-15 mL, ultrafiltered, virus-free water is added and distributed as above.</li>
 <li>The replicate bottles are incubated at in situ conditions using environmental chambers.
 <ol>
 <li>Light levels are altered to surface conditions by using blue-tinted acrylic or clear acrylic with screening net to decrease light intensity.</li>
 <li>Ambient surface temperatures are often obtained by using a flowing seawater deck incubator.</li>
 </ol>
 </li>
 <li>Samples for bacterial and viral abundance estimates are taken at time 0 with a final concentration of 2.0-2.5% sterile glutaraldehyde added into cryovials. These samples are immediately flash frozen with liquid nitrogen and stored frozen until processed.
 <ol>
 <li>If liquid nitrogen is not available, microscopy slides may be prepared and processed immediately (see procedure below)</li>
 </ol>
 </li>
 <li>Subsamples are collected every 2.5 hours for at least 10 hours by the method described above.
 <ol>
 <li> At this time water may be collected for quantitative PCR analysis. Up to 5 mL of the sample may be added to a cryovial with no fixative agent with immediate flash freezing in liquid nitrogen.</li>
 </ol>
 </li>
</ol>
4. Viral Production Microscopy (Noble &amp; Fuhrman 1998, Wen et al. 2004)
<ol>
 <li>Frozen samples to be 0.02-&mu;m filtered for microscopy should be thawed on ice.</li>
 <li>Prepare a stock solution of SYBR Green by diluting the stock solution 1:10 with sterile water. Next, from the stock solution, prepare a working solution by adding 1mL of the stock solution to 39 mL of sterile water. A 50% glycerol, 50% phosphate buffered saline solutions (PBS, 0.05 M Na2HPO4, 0.85% NaCl, pH 7.5) and fresh 2.5% stock solution of p-phenylenediamine should also be prepared before beginning. Keep the 50% glycerol/50% PBS solution at 4&deg;C and the p-phenylenediamine stock at -20&deg;C in the dark until starting. Right before filtering add the p-phenylenediamine to the 50% glycerol/50% PBS to a final concentration of 0.1% to be used as the Antifade solution.</li>
 <li>Place a 25 mm 0.02-&mu;m Anodisc filter on top of a 0.45-&mu;m MicronStep, cellulosic backing filter. Add 850 &mu;L of the fixed sample to the top of the Anodisc and vacuum at 20 pKa until completely dried. Place 100 &mu;L of SYBR Green working solution to a sterile Petri dish and, with the vacuum still on, carefully remove the Anodisc from the filter tower and place on the SYBR Green. Incubate the samples in the dark at room temperature for 20 minutes. Carefully remove the filter from the SYBR Green solution and wick the back of the filter with a Kimwipe to remove all residual dye. If desired, return the filter to the tower and pass up to 800 &mu;L of 0.02-&mu;m filtered water or sterile media through the filter to rinse off excess stain.</li>
 <li>Add a small drop of antifade solution to a microscope slide and place a cover slip on top. Remove the cover slip and add the dried filter to the microscope slide wet with the antifade solution. Again, add a small amount of antifade solution to the cover slip and slowly place it on top of the filter, making sure to get rid of any bubbles that may form.</li>
 <li>Immediately freeze the slides at -20&deg; C until needed (these should be used within a few months to prevent fading and lowered virus counts)</li>
 <li>Viruses are enumerated using fluorescence microscopy (in our case a Leica DMRXA microscope) with a wide blue filter set (&lambda;Ex= 450 to 490 nm, &lambda;Em= 510 nm with a suppression filter at &lambda;= 510 nm). Each filter will have at least 20 fields of view counted, making sure to quantify total viruses from each field grid to ensure even distribution of viruses across the filter membrane.</li>
 <li>Averaged rates of virus reoccurrence from the three independent replicates are then calculated and a standard deviation is determined from the production rates.</li>
</ol>
5. Representative Results
The raw data collected by the researcher requires minimal mathematical processing to generate reoccurrence rates of virus abundance. The primary data set resulting from this study is the reoccurrence rates of virus abundance in the subsamples from the incubations. These results form independent regressions of virus abundance vs time for each of the samples. For each sample the individual incubations act as one treatment, so by completing three replicate-incubations the researcher can calculate rates as well as an estimate of variation (e.g., standard deviation) (see Figure 1.)
One caveat of this process is that the reduction in virus abundance invariable leads to a reduction in the host cells in the sample that are carrying the virus burden. To offset this loss, enumeration of bacterial abundance from both the source (unfiltered seawater or lake water) and the T = 0 incubation sample are necessary. This information can be used to account for the percentage of cells lost: assuming that the process of reducing virus abundance is not selective for or against any members of the microbial community, this factor can then be used to estimate the in situ production rate of viruses.
<img src="/files/ftp_upload/2196/2196fig1.jpg" alt="Figure 1" /> Figure 1. The production of virus-like particles over a 10 hour incubation at in situ conditions using epifluorescence microscopy. Samples were collected during a phytoplankton bloom off the coast of New Zealand in September of 2008. <img src="/files/ftp_upload/2196/2196fig2.jpg" alt="Figure 2" /> Figure 2. Schematic diagram of the work flow process for assaying virus production. The process starts with the ultrafiltration of sample water to generate virus-free water. This is completed using an ultrafiltration system. In parallel water samples are collected from the same site and the free viruses passed through a filter while the microbial community (containing a mixture of infected and non-infected cells) is retained. This community is then resuspended in the virus free water and incubated under in situ conditions. The reoccurrence rate of viruses is then monitored for the next 10 hours to determine rates of virus production.

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+release+of+Fe+and+its+bioavailability+to+marine+plankton.">Viral release of Fe and its bioavailability to marine plankton.</a> Limnol Oceanogr. 49, 1734-1741 .</li><li>Rowe, J. M., Saxton, M. A., Cottrell, M. T., DeBruyn, J. M., Berg, G. M., Kirchman, D. L., Hutchins, D. A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constraints+on+virus+production+in+the+Sargasso+Sea+and+North+Atlantic.">Constraints on virus production in the Sargasso Sea and North Atlantic.</a> Aquat Microb Ecol. 52, 233-244 (2008).</li><li>Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+of+prokaryotic+viruses.">Ecology of prokaryotic viruses.</a> FEMS Microbiol Rev. 28, 127-181 (2004).</li><li>Weinbauer, M. G., Peduzzi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency,+size+and+distribution+of+bacteriophages+in+different+marine+morphotypes.">Frequency, size and distribution of bacteriophages in different marine morphotypes.</a> Mar Ecol Prog Ser. 108, 11-20 (1994).</li><li>Weinbauer, M. G., Winter, C., H&amp;ouml, f. l. e., G, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconsidering+transmission+electron+microscopy+based+estimates+of+viral+infection+of+bacterioplankton+using+conversion+factors+derived+from+natural+communities.">Reconsidering transmission electron microscopy based estimates of viral infection of bacterioplankton using conversion factors derived from natural communities.</a> Aquat Microb Ecol. 27, 103-110 (2002).</li><li>Wen, K., Ortmann, A. C., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+estimation+of+viral+abundance+by+epifluorescence+microscopy.">Accurate estimation of viral abundance by epifluorescence microscopy.</a> Appl Environ Microbiol. 70, 3862-3867 (2004).</li><li>Wilhelm, S. W., Brigden, S. M., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dilution+technique+for+the+direct+measurement+of+viral+production:+a+comparison+in+stratified+and+tidally+mixed+coastal+waters.">A dilution technique for the direct measurement of viral production: a comparison in stratified and tidally mixed coastal waters.</a> Microb Ecol. 43, 168-173 (2002).</li><li>Wilhelm, S. W., Matteson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freshwater+and+marine+virioplankton:+a+brief+overview+of+commonalities+and+differences.">Freshwater and marine virioplankton: a brief overview of commonalities and differences.</a> Freshw Biol. 53, 1076-1089 (2008).</li><li>Wilhelm, S. W., Poorvin, L., Paul, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantification of algal viruses in marine samples. , 53-66 (2001).</li><li>Wilhelm, S. W., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viruses+and+nutrient+cycles+in+the+sea.">Viruses and nutrient cycles in the sea.</a> Bioscience. 49, 781-788 (1999).</li><li>Wilhelm, S. W., Weinbauer, M. G., Suttle, C. A., Jeffrey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+sunlight+in+the+removal+and+repair+of+viruses+in+the+sea.">The role of sunlight in the removal and repair of viruses in the sea.</a> Limnol Oceanogr. 43, 586-592 (1998).</li><li>Winget, D. M., Williamson, K. E., Helton, R. R., Wommack, K. 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No conflicts of interest declared.

A critical component in the understanding how viruses influence marine microbial communities is to determine the rate at which virus particle are produced. Given that abundances are (more or less) static in most systems (Wilhelm &amp; Suttle 1999, Weinbauer 2004), and that viruses are removed or rendered non-infectious quickly in aquatic systems (Wilhelm et al. 1998), then production rates must be relative rapid to replace lost particles. Estimating the mortality viruses cause to the microbial community requires a knowledge of how many viruses are produced every time a virus lyses a cell (the "burst size"). Virus burst sizes in natural samples can vary greatly. Burst sizes can be determined directly by transmission electron microscopy (e.g., Weinbauer &amp; Peduzzi 1994), but this is often beyond the capabilities of a given laboratory or not always practical. In situations where they cannot be empirically determined, literature values of 24 viruses per lytic event may be used for marine systems and 34 for freshwater systems (Parada et al. 2006). If the rate of virus production is divided by this number, the result is the abundance of cells per volume destroyed by viruses on a daily basis. The microbes lysed value can then be divided by the standing stock of bacterial abundance resulting in the virus induced mortality for the system in question: existing estimates range from few percent to nearly the entire population and are often dependent on other factors in the system in question (Wilhelm &amp; Matteson 2008). To determine the percentage of total mortality this number is often multiplied by two (working from the assumption that 50% of the cells go on to reproduce and 50% of the cells are lost, Weinbauer 2004). Given that nutrient and trace element bioavailability (e.g., N, P, Fe) can limit the rate of primary productivity, and as such carbon flux, through aquatic systems, and understanding of the role of virus-driven microbial mortality in this process has become of interest to marine geochemists. Several estimates now exist that suggest viruses release a significant concentrations of nutrient elements back to the water column on a daily basis (Rowe et al. 2008, Higgins et al. 2009) and that these elements are rapidly assimilated by the microbial community (Poorvin et al. 2004, Mioni et al. 2005). The rate of nutrient flux to the environment can be determined by multiplying the number of cells destroyed by the amount of nutrient per cell (denoted "quotas"). This information can provide a critical component to our understanding of how microbial food webs function across aquatic systems. Ongoing developments: Current efforts by a series of research groups involve adapting the above strategy to enumerate specific viruses within the community and, as such, to determine how specific organisms are influenced by virus activity. To do this researchers use the quantitative polymerase chain reaction (qPCR) to estimate the abundance of specific viruses groups or families in parallel to the estimates of the total virus community. The results are then directly applied to provide estimates of virus mortality, nutrient turnover, etc for specific plankton groups. This powerful new approach will allow researchers in the coming years to dig much more deeply into processes associated with the ecology of viruses and, for the first time, to quantify the interactions of specific virus-host communities beyond the constraints of laboratory systems.

<table><tbody><tr><td>2.5% &lt;em&gt;p&lt;/em&gt;-phenylenediamine</td><td>Acros Organics</td><td>130575000</td><td>Stock for Antifade</td></tr><tr><td>Amicon Proflux M12 system</td><td>EMD Millipore</td><td>N/A</td><td>Any ultrafiltration device may be used for this step</td></tr><tr><td>SYBR Green I nucleic acid gel stain</td><td>Invitrogen</td><td>S-7563</td><td></td></tr><tr><td>Helicon S10 30kDa Filter</td><td>EMD Millipore</td><td>CDUF010LT</td><td></td></tr><tr><td>Pelicon XL filters 0.22 &amp;mu;m</td><td>Fisher Scientific</td><td>PXGVPPC50</td><td></td></tr><tr><td>GE 0.2 &amp;mu;m PCTE membrane filters (47mm)</td><td>Fisher Scientific</td><td>09-732-35</td><td></td></tr><tr><td>Millipore Labscale Tangential Flow Filtration System</td><td>EMD Millipore</td><td>XX42LSS11</td><td>Other TFF systems may be used for this step</td></tr><tr><td>0.45 &amp;mu;m Micronstep, Cellulosic, white plain filters (25mm)</td><td>Fisher Scientific</td><td>E04WP02500</td><td></td></tr><tr><td>GE Whatman 0.02 &amp;mu;m Anodisc filters (25mm)</td><td>Fisher Scientific</td><td>68-09-6002</td><td></td></tr><tr><td>Leica DMRXA microscope</td><td></td><td></td><td>Any epifluorescence microscope with a blue filter set may be used</td></tr><tr><td>20L polycarbonate carboys</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>BP229-4</td><td></td></tr><tr><td>PBS (0.05 M Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.85% NaCl, pH 7.5)</td><td>Fisher Scientific</td><td>BP329-500, S640-500</td><td></td></tr><tr><td>50% Glutaraldehyde </td><td>Fisher Scientific</td><td>G151-1</td><td></td></tr><tr><td>Corning 2 mL Cryovials-External Thread polypropylene</td><td>Fisher Scientific</td><td>09-761-71</td><td>Any cryovials may be used</td></tr><tr><td>Corning 5 mL Cryovials-External Thread polypropylene</td><td>Fisher Scientific</td><td>09-761-74</td><td>Any cryovials may be used</td></tr><tr><td> 85% H&lt;sub&gt;3&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>A242-212</td><td>Spiral Membrane storage</td></tr><tr><td>NaOH pellets</td><td>Fisher Scientific</td><td>S318-3</td><td>M12 cleaning</td></tr><tr><td>Graduated Cylinders</td><td></td><td></td><td></td></tr><tr><td>Isopore 0.8-&amp;mu;m pore-size membrane filter (142mm)</td><td>EMD Millipore</td><td>ATTP14250</td><td></td></tr><tr><td>Millipore Stainless Steel Pressure Filter Holder (142mm)</td><td>Fisher Scientific</td><td>YY30 090 00</td><td></td></tr></tbody></table>

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.

Watch this Scientific Journal Video about Estimating Virus Production Rates in Aquatic Systems at JoVE.com

Estimating Virus Production Rates in Aquatic Systems

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 ...

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Immunology and Infection

12.6K Views.  University of Tennessee. 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. 

Video: Estimating Virus Production Rates in Aquatic Systems

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.

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 ...

Environment

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

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. ...

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 ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

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

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Monitoring Influenza Virus Survival Outside the Host Using Real-Time Cell Analysis

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are either time-consuming or unable to detect small variations. Presented here is a protocol for the precise quantification of viral titer by analyzing electrical impedance variations of infected cells in real-time. Cellular impedance is measured through gold microelectrode biosensors located under the cells in microplates, in which magnitude depends on the number of cells as well as their size and shape. This protocol allows real-time analysis of cell proliferation, viability, morphology and migration with enhanced sensitivity. Also provided is an example of a practical application by quantifying the decay of influenza A virus (IAV) submitted to various physicochemical parameters affecting viral infectivity over time (i.e., temperature, salinity, and pH). For such applications, the protocol reduces the workload needed while also generating precise quantification data of infectious virus particles. It allows the comparison of inactivation slopes among different IAV, which reflects their capacity to persist in given environment. This protocol is easy to perform, is highly reproducible, and can be applied to any virus producing cytopathic effects in cell culture.

Reported here is a protocol for the quantification of infectious viral particles using real-time monitoring of electrical impedance of infected cells. A practical application of this method is presented by quantifying influenza A virus decay under different physicochemical parameters mimicking environmental conditions.

Methods for virus particle quantification represent a critical aspect of many virology studies. Although several reliable techniques exist, they are ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...