We evaluated a novel, magnetic-bead-based histo-blood group antigen assay for the recovery of low numbers of norovirus particles. Using this assay, with Norwalk virus seeded in environmental waters as a model, we were able to recover 30 to 300 genomic copies of the virus

Cannon, J. L.

Vinje, J.

Carolina Digital Repository

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2008, p. 6818–6819 Vol. 74, No. 210099-2240/08/$08.000 doi:10.1128/AEM.01302-08Copyright © 2008, American Society for Microbiology. All Rights Reserved.Histo-Blood Group Antigen Assay for Detecting Noroviruses in WaterJennifer L. Cannon1,2,3* and Jan Vinjé2Department of Environmental Science and Engineering, University of North Carolina, Chapel Hill, North Carolina1;Gastroenteritis and Respiratory Viruses Laboratory Branch, Division of Viral Diseases, Centers for Disease Control andPrevention, Atlanta, Georgia2; and Atlanta Research and Education Foundation, Decatur, Georgia3Received 11 June 2008/Accepted 28 August 2008We evaluated a novel, magnetic-bead-based histo-blood group antigen assay for the recovery of low numbersof norovirus particles. Using this assay, with Norwalk virus seeded in environmental waters as a model, we wereable to recover 30 to 300 genomic copies of the virus.In recent years, human noroviruses (NoVs) have been rec-ognized as the leading cause of outbreaks of acute gastroen-teritis worldwide. Large NoV outbreaks have been reported inassociation with water used for drinking, recreation, shellfishharvesting, irrigation, and washing produce (1, 2, 3, 5, 7, 10, 13,16, 20). NoVs recognize human histo-blood group antigens(HBGAs) as receptors (4, 12, 14). Since NoVs cannot be cul-tured in vitro, the virus is detected by reverse transcription-PCR (RT-PCR) assays, which are susceptible to inhibitorsoften found in environmental waters. Here, we report a novelmethod for the rapid recovery of low numbers of NoVs by theuse of a magnetic-bead-based HBGA assay.HBGA binding assay optimization. Using a previously de-scribed HBGA binding assay (9) we evaluated three blockingbuffers (5% [wt/vol] Blotto, 5% [vol/vol] fetal bovine serum,and SuperBlock [Pierce Biotechnology, Rockford, IL]) fortheir ability to block nonspecific binding to uncoated magneticbeads without interfering with specific binding of virus toHBGA or interfering with the reproducibility of results. Basedon these criteria, SuperBlock blocking buffer was chosen forsubsequent experiments. In brief, 25 l of washed MyOnestreptavidin-coated magnetic beads (Dynal Biotech, Olso, Nor-way) was incubated for 90 min at room temperature with 50 lof synthetic biotinylated H type 1 HBGA (1 mg/ml) (Glyco-Tech, Rockville, MD), followed by overnight incubation withblocking buffer. RNA copy numbers of the Norwalk virus (NV)stool suspensions were determined by comparison to a stan-dard curve, using NV strain 8FIIb RNA transcripts. Dilutionsof 10% stool suspensions containing 3,000, 300, 30, or 3 NV(8FIIb) copy numbers were added to 1 ml of blocking buffer,environmental water concentrate, or phosphate-buffered salineprior to incubation (4 h at room temperature) on an end-over-end rotator (Dynal Biotech). After eight washes, the beadswere suspended in 50 l of phosphate-buffered saline andsubjected to heat (5 min at 99°C) to release the viral RNA. Forthe HBGA assay, 2.5 l or 1 l of heat-released NV RNA wasanalyzed by use of a conventional RT-PCR (Qiagen OneStepRT-PCR kit; Qiagen, Valencia, CA) (6) or a TaqMan real-time RT-PCR (QuantiTect probe RT-PCR kit; Qiagen) on anApplied Biosystems 7500 real-time PCR system platform (Fos-ter City, CA) (22). The detection limit for the real-time assaywas 10 RNA copy numbers.The method is sensitive and specific for detecting NoV. Amedian of 300 copy numbers (n  10) per milliliter of blockingbuffer was detected by the HBGA assay (Table 1). In thepresence of other enteric viruses (rotavirus group A serotype 1[strain WA] or human astrovirus type 1 [Oxford strain]), 300NV copy numbers were recovered by the assay (n  2).The method is sensitive in the context of environmentalwaters. Surface water (n  4) and influent (n  2) and effluent(n  4) wastewater samples were collected from drinking-water and wastewater facilities in Columbus and Atlanta, GA,respectively. Surface water samples were concentrated by pre-cipitation with 8% polyethylene glycol (PEG) 8000 and 0.3 MNaCl (23). After centrifugation (7,280  g, 30 min), the PEGpellet was extracted with 50% (vol/vol) chloroform. Superna-tants (5 to 20 ml) were stored at 70°C until use. The waste-water samples (850 ml) were processed similarly; only an ad-ditional centrifugation step (7,280  g, 15 min at 4°C) andchloroform extraction of the pellet (50% [vol/vol]) were per-formed prior to PEG precipitation of the combined aqueousphases. Water concentrates were seeded with 3, 30, 300, or3,000 copy numbers of NV and analyzed, using the HBGAbinding assay. The detection limit for each water type testedwas between 30 and 3,000 (median, 300) (Table 1).The method detects viruses with an intact capsid. To deter-mine if the NV particles were intact and potentially infectious,we bound 30,000 or 6,000 NoV copy numbers to H type 1HGBA-coated beads before treatment with 10 ng of RNase A(Invitrogen, Carlsbad, CA) in Tris-EDTA buffer and incuba-tion for 30 min at 37°C. To neutralize any remaining RNaseactivity, RNase inhibitor (160 U/50 l sample) (Invitrogen)was added, and the sample was incubated for 15 min at 37°C.Before and after RNase treatment, GII.4 RNA (1  105 copynumbers) was added to each reaction mixture to check theefficacy of the RNase A or the RNase inhibitor. The meancycle threshold values for the treated (35.8; range, 34.5 to 37.0)and untreated (35.2; range, 34.5 to 35.9) samples did not differsignificantly (P  0.2; Student’s t test) at either input level. Asexpected, RNase treatment of the GII.4 control RNA resultedin complete loss of signal, when analyzed by TaqMan real-timeRT-PCR (22).* Corresponding author. Present address: University of Georgia,Center for Food Safety, 1109 Experiment St., Melton Bldg., Griffin,GA 30223. Phone: (770) 467-6094. Fax: (770) 229-3216. E-mail:jcannon@uga.edu. Published ahead of print on 5 September 2008.6818In summary, we report a novel HBGA magnetic bead sep-aration method for human NoVs. The method is sensitive andspecific for detecting NoVs with an intact HBGA receptorbinding site. Furthermore, since we demonstrated only a 1-logdecrease in method sensitivity upon application to sewage sam-ples, this assay can be used to remove RT-PCR inhibitorycompounds present in environmental waters.Several research groups have developed sensitive methodsfor concentrating NoVs by use of immunomagnetic separationor gastric mucins from pigs (8, 17, 18, 19, 21). Our method candetect low numbers of NoVs, but it also detects NoV bound toits specific HBGA receptor, which may be a surrogate fordetecting infectious virus. Previous studies indicate that many,but not all, NoVs bind to HBGAs (9, 11, 15), and the require-ment of a secondary receptor during infection is not clear.Elucidating the role of HBGA and NoV infectivity will beneeded to further validate the value of this assay as a surrogatefor detecting infectious virus. In conclusion, we report an assaythat may serve as a rapid detection method for potentiallyinfectious NoVs in complex matrices, such as environmentalwaters.We thank Christine Moe (Emory University) and Robert Atmar(Baylor College of Medicine) for kindly providing Norwalk virusstocks, Linda Vermeulen for her assistance with the RNase A exper-iments, and Claudia Chesley for critical reading of the manuscript.This project was supported by American Water Works and ResearchFoundation project no. 2860.The findings and conclusions in this article are those of the authorsand do not necessarily represent the views of the Centers for DiseaseControl and Prevention (CDC). This article did receive clearancethrough the appropriate channels at the CDC prior to submission.REFERENCES1. Berg, D. E., M. A. Kohn, T. A. Farley, and L. M. McFarland. 2000. Multi-state outbreaks of acute gastroenteritis traced to fecal-contaminated oystersharvested in Louisiana. J. Infect. Dis. 181:S381–S386.2. Borchardt, M. 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Isolation and characterization of circulatingtype 1 vaccine-derived poliovirus from sewage and stream waters in Hispani-ola. J. Infect. Dis. 189:1168–1175.TABLE 1. Detection limits of the HBGA binding assay for Norwalkvirus in the presence of an environmental water matrix orSuperBlock blocking bufferType ofenvironmental wateror controlNo. of samples detected/total no. of samples ateach indicated input level (RNA copy no.)3,000 300 30 3SuperBlock control 10/10 9/10 4/10 1/10Surface water 4/4 4/4 4/4 0/4Effluent wastewater 4/4 4/4 0/4 0/4Influent wastewater 2/2 1/2 0/2 0/4VOL. 74, 2008 HBGA BINDING ASSAY FOR NoV IN ENVIRONMENTAL WATERS 6819

Histo-Blood Group Antigen Assay for Detecting Noroviruses in Water

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Histo-Blood Group Antigen Assay for Detecting Noroviruses in Water

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