This work was supported by NSERC Alliance Covid-19 Grant (Award No. 431401363, 2020-2021, Drs. Yuan and Uyaguari-D&#237;az). MUD would like to thank the University Research Grants Program (Award No. 325201). Both JF and JZA are supported by the Visual and Automated Disease Analytics (VADA) graduate training program. KY and JF both received fellowships from the Mitacs Accelerate program. MUD and his laboratory members (KY, JF, JZA) are supported by NSERC-DG (RGPIN-2022-04508) and the Research Manitoba New Investigator Operating grant (No 5385). Special thanks to the City of Winnipeg, Manitoba. This research was conducted at the University of Manitoba. We would like to acknowledge that the University of Manitoba campuses are located on the original lands of Anishinaabeg, Cree, Oji-Cree, Dakota, and Dene peoples and on the homeland of the M&#233;tis Nation.

1. Comparison of serial ultrafiltration and skimmed milk flocculation to concentrate viruses in wastewater samples
<ol>
	<li>Sample preparation
	<ol>
		<li>Collect 2 L of 24 h flow-proportional composite raw (influent) wastewater samples. Samples were collected from the three major wastewater treatment plants (WWTPs) in Winnipeg, Canada, during the summer and fall of 2020 (Table 1).</li>
		<li>Transport the samples to the laboratory in light-proof bottles in an icebox and process them within 24 h. Collect wastewater physico-chemical and biological characteristics data.</li>
	</ol>
	</li>
	<li>Virus concentration assays 
	NOTE: A sequential ultrafiltration method and an organic flocculation method, namely skimmed milk flocculation (SMF), were applied to assess the total recovery efficiency of each method using Armored RNA as a test virus23. Other researchers have also used Armored RNA as a control virus for assessing the recovery of concentration methods for enveloped viruses, such as the family Coronaviridae22,24,25,26.
	<ol>
		<li>For the addition of Armored RNA, set the wastewater test volumes for the ultrafiltration and SMF methods as 140 mL and 500 mL, respectively. Using a pipettor, spike 5 x 104 copies of Armored RNA into six fresh wastewater samples collected on one date and six fresh wastewater samples on another date, for each ultrafiltration method; further, spike six stored (at 4 &#176;C) wastewater samples collected on a third, later date and processed days later for SMF. 
		NOTE: Each of the six sample sets collected on different dates was composed of two samples from each of the three WWTPs. In our study, the first set of fresh wastewater samples were collected on July 8th, 2020, the second on September 30th, 2020, and the wastewater samples to be stored for SMF on October 14th, 2020.</li>
		<li>Stir for 30 min at 4 &#176;C to inoculate and homogenize the Armored RNA in the samples using a magnetic stirrer.</li>
		<li>Remove all particles or cells larger than 0.2 &#956;m and perform ultrafiltration.
		<ol>
			<li>Filter the spiked wastewater samples (120 mL from each 140 mL spiked sample) through cheesecloth and low-protein binding, 0.45 &#181;m and 0.2 &#181;m, 47 mm membrane disc filters, respectively, to remove large particles, sediments, eukaryotes, and bacteria27. Process the filtered samples using the ultrafiltration methods described below. 
			NOTE: The wastewater samples collected on July 8th and September 30th were used for ultrafiltration methods with 3,000 x g and 7,500 x g, respectively.</li>
			<li>For sequential ultrafiltration at 3,000 x g (UF-3k x g), concentrate a total of 120 mL of the test sample collected on the first date at 3,000 x g for 30 min by loading 60 mL of the sample twice to approximately 5 mL using a brand A centrifugal device, 30-kDa. Then, concentrate down the 5 mL volume at 3,000 x g for 30 min using a brand B centrifugal device, 30-kDa, to 500-1,200 &#181;L.</li>
			<li>For sequential ultrafiltration at 7,500 x g (UF-7.5k x g), change the centrifugation speed for the brand B centrifugal device from 3,000 x g to 7,500 x g to obtain smaller final volumes, as per the manufacturer&#39;s recommendations, and keep the centrifugation speed of the brand A centrifugal device the same. 
			NOTE: The tests were run with the samples collected on September 30th.</li>
		</ol>
		</li>
		<li>For SMF, perform the steps outlined below.</li>
		<li>Spike the wastewater samples (500 mL each) to directly concentrate using the SMF protocol16,18, without prefiltration with cheesecloth and low-protein binding membrane disc filters, as described below.</li>
		<li>Dissolve 0.5 g of skimmed milk powder in 50 mL of synthetic seawater to obtain a 1% (w/v) skimmed milk solution and carefully adjust the pH of the solution to 3.5 using 1 N HCl. 
		NOTE: Acidification makes viruses aggregate and precipitate out of water due to the change in their isoelectric point16 and improves the solubility of milk powder28.</li>
		<li>Add 5 mL of skimmed milk solution to 500 mL of raw wastewater samples to obtain a final concentration of skimmed milk of 0.01% (w/v).</li>
		<li>Stir the samples for 8 h at medium speed and allow the formed flocs to settle for another 8 h at room temperature.</li>
		<li>Remove the supernatants carefully using serological pipettes without disturbing the settled flocs. Transfer a final volume of 50 mL containing the flocs to centrifuge tubes and centrifuge at 8,000 x g for 30 min at 8 &#176;C.</li>
		<li>Carefully scrap the pellets using a sterilized spatula, and resuspend the remaining pellets in the tubes in 250 &#181;L of 0.2 M sodium phosphate buffer (pH 7.5) after the supernatant is removed. Transfer the scraped and resuspended pellets to the same 1.5 mL mini-centrifuge tubes.</li>
	</ol>
	</li>
	<li>Perform viral RNA extraction from viral concentrates of wastewater samples and recovery efficiency assays using an RNA extraction kit with a 25:24:1 ratio of phenol:chloroform:isoamyl alcohol and &#946;-mercaptoethanol, according to the manufacturer&#39;s instructions, to improve the extraction efficiency. Finally, elute the RNA in 50 &#181;L of elution buffer.</li>
	<li>Real time-quantitative polymerase chain reaction (RT-qPCR) analysis
	<ol>
		<li>Quantify Armored RNA using tenfold dilutions (7.8 x 104 to 7.8 gene copies) of a synthetic single-stranded DNA or gBlock construct. See Table 2 for the primers and probe sets that detect and quantify the Armored RNA.</li>
		<li>Design primer and probe sets using the primer design tool29 and target a 95 bp region (gBlock construct) within the Armored RNA genome.</li>
		<li>Obtain calibration curves for each RT-qPCR run. Include negative controls in each qPCR run. Run standards, samples, and non-template controls in triplicate.</li>
		<li>Prepare each 10 &#181;L RT-qPCR mixture in the following order: 2.5 &#181;L of 4x master mix, 400 nM of each primer, 200 nM probe, and 2.5 &#181;L of template, as well as ultrapure DNAse/RNAse- free distilled water.</li>
		<li>Perform thermal cycling reactions at 50 &#176;C for 5 min, followed by 45 cycles of 95 &#176;C for 10 s and 60 &#176;C for 30 s on a real-time PCR system. If the CT was &#60;40, consider the sample Armored RNA positive.</li>
		<li>Conduct qPCR and RT-qPCR tests using bovine serum albumin with raw sewage samples to assess the possibility of inhibitors or contaminants such as humic acids24. No significant differences were observed between samples with and without the enzyme.</li>
	</ol>
	</li>
	<li>Calculate the concentrations from the standard curve. Calculate the recovery efficiency percentage by dividing the recovered concentration by the spiked concentration.</li>
	<li>Transform gene copy numbers using the log10 function for analysis. Run a general linear model using a statistical analysis tool on the qPCR data. Apply Tukey&#39;s test to detect differences among the methods and treatments. Take a p-value of 0.05 to be a minimum significance level for the test.</li>
</ol>
2. Filtration and concentration of DNA and RNA viruses for water-based epidemiology
<ol>
	<li>For environmental surface water collection, collect 10 L of environmental surface water samples and use 10 L of deionized water samples for background control. Store all the samples in a 4 &#176;C room until processing within 24 h following sample collection.</li>
	<li>Perform concentration of viruses from environmental samples with SMF using the steps described below.
	<ol>
		<li>Perform capsule and vacuum filtration using&#160;high-capacity groundwater sampling capsules and membrane disc filters of 0.45 &#181;m, 0.2 &#181;m, and 0.1 &#181;m sizes to minimize noise during metagenomic sequencing, from larger fractions such as micro-eukaryotes and bacteria to smaller ones such as viruses.</li>
		<li>Prepare a 1% weight-by-volume pre-flocculated skimmed milk solution in 1.32 L of synthetic seawater with 13.2 g of skimmed milk powder. Adjust the pH of this suspension to 3.5 using 1 N HCl.</li>
	</ol>
	</li>
	<li>Adjust the pH of the environmental surface water samples to 3.5 using 1 N HCl.</li>
	<li>Transfer 100 mL of the pre-flocculated acidified skimmed milk solution to 10 L acidified (pH 3.5) environmental water samples (final skimmed milk concentration of 0.01% [w/v]).</li>
	<li>Using a magnetic stirrer and a magnetic stir bar, stir the samples for 8 h at room temperature and allow the flocs to sediment by gravity for an additional 8 h. Without disturbing the flocs, carefully remove the supernatant using a vacuum pump.</li>
	<li>Aliquot and balance the remaining flocs according to the weight in 50 mL centrifuge tubes and spin them down at 8,000 x g for 30 min at 4 &#176;C.</li>
	<li>Discard the supernatant and dissolve the resulting pellet (which theoretically contains viruses of interest) in 200 &#181;L of 0.2 M sodium phosphate buffer.</li>
	<li>Treat the dissolved pellet with DNase I and RNase A, following the manufacturer&#39;s instructions, to eliminate free DNA and RNA present that may be co-precipitated owing to the kit used for nucleic acid extraction. Inactivate the DNase I and RNase A following the manufacturer&#39;s instructions.</li>
	<li>Extract total nucleic acids from the dissolved pellet using a DNA/RNA extraction kit, as per the manufacturer&#39;s instructions.</li>
	<li>Quantify the total amount of extracted DNA and RNA from effluent samples using a fluorometer. Samples with concentrations &#62;1 ng/&#181;L are considered optimal for DNA quantification and sequencing.</li>
	<li>Perform qPCR analysis to target enteric viruses of interest and high-throughput sequencing to identify the viral community structure.</li>
</ol>
3. Direct precipitation and concentration of microbial fractions for water-based epidemiology
<ol>
	<li>Collect 2 L of water samples from protected and impacted waterways. If the sample contains a considerable amount of debris, use a cheesecloth to remove them. 
	NOTE: Collecting a biological duplicate per sample is highly recommended.</li>
	<li>Prepare a 1% (w/v) skimmed milk solution by dissolving 4.4 g of skimmed milk powder in 440 mL of synthetic seawater. Adjust the pH of this suspension to 3.5 with 1 N HCl.</li>
	<li>Adjust the pH of the freshwater samples to 3.5 using 1 N HCl.</li>
	<li>Add 20 mL of skimmed milk solution to the previously pH-adjusted 2 L freshwater samples (final skimmed milk concentration of 0.01% [w/v]). Stir the samples at room temperature for 8 h.</li>
	<li>Allow the flocs to sediment by gravity for another 8 h. Carefully remove the supernatants with a peristaltic pump. Transfer the sedimented flocs to a 50 mL centrifuge tube and spin them down at 8,000 x g for 30 min.</li>
	<li>Discard the supernatant and resuspend the pellets with 200 &#181;L of 0.2 M sodium phosphate buffer. Store the samples at -20 &#176;C or proceed to select ~ 0.5 g of the floc for microbial DNA extraction by using the nucleic acid isolation kit of choice, following the manufacturer&#39;s instructions.</li>
	<li>Determine the DNA nucleic acid concentration and purity with a high sensitivity fluorometer. Samples with concentrations &#62;1 ng/&#181;L should be optimal for DNA quantification and sequencing.</li>
</ol>

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

One of the critical steps in this study is the elimination of solid particles by applying a prefiltration step with 0.2 &#181;m and 0.45 &#181;m membrane filters. Considering the partitioning of viruses into solid particles, especially enveloped viruses, prefiltration can cause a significant loss in viral recovery30. While a prefiltration step for ultrafiltration methods is almost always necessary for environmental and wastewater samples to prevent ultrafiltration devices from clogging, prefiltrat...

Determining the effective concentration of microorganisms in surface and wastewater samples for microbial community analysis&#160;and&#160;epidemiology studies, is one of the important steps for monitoring and predicting the course of outbreaks in communities1,2. The COVID-19 pandemic, unfolded the importance of improving concentration methods. COVID-19 emerged in late 2019 and, as of March 2023, still poses a threat to human health, social life, and the economy. Effective surveillance and control strategies to alleviate the impacts of COVID-19 outbreaks in communities have become an important research topic, as new waves and variants of COVID-19 have been emerging in addition to the rapid transmission and spread of the virus, as well as unreported and undiagnosed asymptomatic cases3,4,5. The use of wastewater-based epidemiology for COVID-19 by civil society organizations, government agencies, and public or private utilities has been helpful in providing rapid outbreak-related information and mitigating the impacts of COVID-19 outbreaks6,7,8,9. However, the concentration of SARS-CoV-2, an enveloped RNA virus, in wastewater samples still poses challenges10. For example, one of these challenges is the partitioning of SARS-CoV-2 in wastewater solids, which may impact recovery when the solids are eliminated during concentration11. If this is the case, the focus of quantification/assessment should be on both solid and aqueous phases of environmental water samples, rather than the aqueous phase only. Furthermore, the choice of concentration method can be modified based on downstream tests and analyses. The concentration of virus particles and pathogens from environmental samples has become an urgent research topic with developments in sequencing and microbiome fields.
Various virus concentration methods have been applied in the field of virus concentration from environmental water and wastewater samples. Some commonly used methods are filtration, skimmed milk flocculation (SMF), adsorption/elution, and polyethylene glycol precipitation12-17. Among them, SMF has been considered a cheap and effective method, successfully tested, and applied for recovering viruses, including SARS-CoV-2, from wastewater and surface waters12,15,16,18. The SMF procedure is a relatively new approach that has gained increased recognition among many environmental studies as an appropriate methodology to simultaneously recover a broad array of microorganisms such as viruses, bacteria, and protozoans from all types of water samples, namely sludge, raw sewage, wastewater, and effluent samples19. When compared to other known methodologies to recover viruses from environmental samples such as ultrafiltration and glycine-alkaline elution, lyophilization-based approach, or ultracentrifugation and glycine-alkaline elution, SMF has been reported as the most efficient method with higher viral recovery and detection rates18,20. In the present study, we used Armored RNA as a test virus to assess the recovery efficiency of virus concentration methods, including tests for assessing SARS-CoV-2 recovery21,22.
Here, we tested wastewater and environmental water samples to demonstrate the utility of SMF and a sequential ultrafiltration method to concentrate microbial fractions for quantitative polymerase chain reaction (qPCR), sequence-based metagenomics, and deep-amplicon sequencing. SMF is a relatively cheaper method and optimal for a larger volume of samples compared to ultrafiltration methods. 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.

<table><tbody><tr><td>0.2 M sodium phosphate buffer with a pH 7.5</td><td>Alfa Aesar</td><td>J62041AP</td><td>Fisher Scientific, Fair Lawn, NJ, USA</td></tr><tr><td>0.2 &amp;mu;m 47-mm Supor-200 membrane disc filters</td><td>VWR</td><td>66234</td><td>Pall Corporation, Ann Arbor, MI</td></tr><tr><td>0.45 &amp;mu;m 47-mm Supor-200 membrane disc filters</td><td>VWR</td><td>60043</td><td>Pall Corporation, Ann Arbor, MI</td></tr><tr><td>4X TaqMan Fast Virus 1-Step Master Mix</td><td>Thermo Fisher Scientific</td><td>4444432</td><td>Life Technologies, Carlsbad, CA, USA</td></tr><tr><td>Armored RNA Quant IPC-1 Processing Control</td><td>Asuragen</td><td>49650</td><td>Asuragen, Austin, TX, USA</td></tr><tr><td>Brand A, Jumbosep Centrifugal Device, 30-kDa</td><td>Pall&amp;nbsp;</td><td>OD030C65</td><td>Pall Corporation, Ann Arbor, MI</td></tr><tr><td>Brand B, Microsep Advance Centrifugal Device, 30-kDa</td><td>Pall</td><td>MCP010C46</td><td>Pall Corporation, Ann Arbor, MI</td></tr><tr><td>Centrifuge tubes (50 ml)&amp;nbsp;</td><td>Nalgene</td><td>3119-0050PK</td><td>Thermo Fisher Scientific</td></tr><tr><td>DNAse I</td><td>Invitrogen</td><td>18047019</td><td>Thermo Fisher Scientific</td></tr><tr><td>Dyna Mag-2</td><td>Invitrogen</td><td>12027</td><td>Thermo Fisher Scientific</td></tr><tr><td>GWV High Capacity Groundwater Sampling Capsules - 0.45 &amp;micro;m</td><td>Pall</td><td>12179</td><td>Pall Corporation, Ann Arbor, MI</td></tr><tr><td>Hydrochloric acid, 1N standard solution</td><td>Thermo Fisher Scientific</td><td>AC124210025</td><td>Fisher Scientific, Fair Lawn, NJ, USA</td></tr><tr><td>MagMAX Microbiome Ultra Nucleic Acid Isolation Kit</td><td>Applied biosystems</td><td>A42358</td><td>Thermo Fisher Scientific</td></tr><tr><td>Nuclease free water</td><td>Promega</td><td>P1197</td><td>Promega Corporation, Fitchburg, WI, USA</td></tr><tr><td>Peristaltic pump</td><td>Masterflex, Cole-Parmer instrument</td><td>7553-20</td><td>Thermo Fisher Scientific</td></tr><tr><td>pH meter&amp;nbsp;</td><td>Denver instrument</td><td>RK-59503-25</td><td>Cole-Parmer. This product has been discontinued</td></tr><tr><td>Phenol:chloroform:isoamyl alcohol 25:24:1</td><td>Invitrogen</td><td>15593031</td><td>Fisher Scientific, Fair Lawn, NJ, USA</td></tr><tr><td>Primers and probe sets</td><td>IDT</td><td></td><td>Integrated DNA Technologies, Inc., Coralville, IA, USA</td></tr><tr><td>Qiagen All-prep DNA/RNA power microbiome kit</td><td>Qiagen</td><td></td><td>Qiagen Sciences, Inc., Germantown, MD, USA</td></tr><tr><td>QuantStudio 5 Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td>A34322</td><td>Life Technologies, Carlsbad, CA, USA</td></tr><tr><td>Qubit 1X dsDNA High Sensitivity (HS) assay kit</td><td>Invitrogen</td><td>Q33231</td><td>Thermo Fisher Scientific</td></tr><tr><td>Qubit 4 Fluorometer, with WiFi</td><td>Invitrogen</td><td>Q33238</td><td>Thermo Fisher Scientific</td></tr><tr><td>Qubit RNA High Sensitivity (HS) assay kit</td><td>Invitrogen</td><td>Q32855</td><td>Thermo Fisher Scientific</td></tr><tr><td>RNAse A</td><td>Invitrogen</td><td>EN0531</td><td>Thermo Fisher Scientific</td></tr><tr><td>RNeasy PowerMicrobiome Kit</td><td>Qiagen</td><td>26000-50</td><td>Qiagen Sciences, Inc., Germantown, MD, USA</td></tr><tr><td>Skim milk powder</td><td>Difco (BD Life Sciences)</td><td>DF0032173</td><td>Fisher Scientific, Fair Lawn, NJ, USA</td></tr><tr><td>Sodium phosphate buffer</td><td>Alfa Aesar</td><td></td><td>Alfa Aesar, Ottawa, ON, Canada</td></tr><tr><td>Synthetic seawater</td><td>VWR&amp;nbsp;</td><td>RC8363-1</td><td>RICCA chemical company</td></tr><tr><td>Synthetic single-stranded DNA gBlock</td><td>IDT</td><td></td><td>Integrated DNA Technologies, Inc., Coralville, IA, USA</td></tr><tr><td>VacuCap 90 Vacuum Filtration Devices - 0.1 &amp;micro;m, 90 mm, gamma-irradiated</td><td>Pall</td><td>4621</td><td>Pall Corporation, Ann Arbor, MI</td></tr><tr><td>VacuCap 90 Vacuum Filtration Devices - 0.2 &amp;micro;m, 90 mm, gamma-irradiated</td><td>Pall</td><td>4622</td><td>Pall Corporation, Ann Arbor, MI</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Gibco</td><td>21985023</td><td>Fisher Scientific, Fair Lawn, NJ, USA</td></tr></tbody></table>

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.

Evaluation of viral RNA concentration methods 
All six samples processed with UF-3k x g were positive and resulted in a 13.38% &#177; 8.14% recovery (Figure 1). Only one sample was positive when the samples were processed with UF-7.5k x g. All samples processed with SMF were positive and resulted in a 15.27% &#177; 2.65% recovery (Figure 1). The average recovery rates of UF-3K x g and SMF were significantly and co...

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

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 ...

concentration-virus-particles-from-environmental-water-wastewater

Research

JoVE Journal

Environment

1.4K Views.  University of Manitoba. Epidemiologie op basis van water en afvalwater is naar voren gekomen als een alternatieve methode om het verloop van uitbraken in gemeenschappen te volgen en te voorspellen. De mageremelkflocculatiemethode is een alternatieve benadering van concentratiemicroben. Magere melk flocculatiemethode vertegenwoordigt een veelzijdige, goedkope en gemakkelijke hands-off methode om microbiële fracties te concentreren zonder significante verschillen in vergelijking met ultrafiltratie of tangentiële str...

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

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 ...

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

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. 
Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. 
This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.

Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields &#62; 6 &#215; 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described.

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both ...

Cancer Research

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 ...

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 ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Growth, Purification, and Titration of Oncolytic Herpes Simplex Virus

Oncolytic viruses (OVs), such as&#160;the&#160;oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer immunotherapy. OVs, including oHSV, selectively replicate in and kill cancer cells (sparing healthy/normal cells) while inducing anti-tumor immunity. Because of these unique properties, oHSV-based treatment strategies are being increasingly used for the treatment of cancer, preclinically and clinically, including FDA-approved talimogene laherparevec (T-Vec). Growth, purification, and titration are three essential laboratory techniques for any OVs, including oHSVs, before they can be utilized for experimental studies. This paper describes a simple step-by-step method to amplify oHSV in Vero cells. As oHSVs multiply, they produce a cytopathic effect (CPE) in Vero cells. Once 90-100% of the infected cells show a CPE, they are gently harvested, treated with benzonase and magnesium chloride (MgCl2), filtered, and subjected to purification using the sucrose-gradient method. Following purification, the number of infectious oHSV (designated as plaque-forming units or PFUs) is determined by a &#34;plaque assay&#34; in Vero cells. The protocol described herein can be used to prepare high-titer oHSV stock for in vitro studies in cell culture and in vivo animal experiments.

In this manuscript, we describe a simple method of growth, purification, and titration of the oncolytic herpes simplex virus for preclinical use.

Oncolytic viruses (OVs), such as&#160;the&#160;oncolytic herpes simplex virus (oHSV), are a rapidly growing treatment strategy in the field of cancer ...

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

Biology

AAV Isolation by Double Iodixanol Density Gradient Centrifugation

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

Author Spotlight: Efficient Adeno-Associated Virus Isolation for Pre-Clinical Applications

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a ...

Medicine

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

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

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