A Small Volume Procedure for Viral Concentration from Water

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

1. Preparation of Glassware and Filter Housings

1. Unless otherwise noted, sterilize all glassware, filter housings and solutions at 121 °C for 15 min. To ensure sterility, cover all openings or exposed surfaces with either aluminum foil or tape-secured paper prior to sterilization.
2. 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.
3. Prepare 1 L of 1.5% beef extract (flocculating; produces floc particles when pH of solution is lowered to 3.5, or non-flocculating; whi....

Results

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% ± 52% (fine), 115% ± 28% (medium) and 82% ± 53% (large) particle celites and for AdV41, 132% ± 39% (fine), 83% ± 25% (medium) and.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adenovirus 40 stock	ATCC	VR-931
Adenovirus 41 stock	ATCC	VR-930
Sodium Thiosulfate	Fluka Chemical Co.	72051
Celites #577	Fluka Chemical Co.	22142
NanoCeram 47 mm	Argonide	N/A
1MDS 47 mm	3M	6408502
AP-20 Prefilter 47 mm	Millipore Corp.	AP2004700
Glycine	Sigma	50046-1KG
Sodium Polyphosphate	Acros Organics	390930010
Trypsin	Gibco	25200
PBS	Sigma	P5368
Hydrochloric Acid	Fisher	A481-212
BBL Beef Extract	BD Biosciences	212303
Difco Beef Extract	BD Biosciences	211520
ABI 7900 Real-time PCR system	ABI	N/A
Stainless Steel Filter Housing	Millipore Corp.	XX2004720
Blood DNA Extraction Kit	Qiagen	51104
EPA MPN Calculator			http://www.epa.gov/nerlcwww/online.html