Blanca I. Escudero-Abarca

Outbreak of Norovirus Infection among River Rafters Associated with Packaged Delicatessen Meat, Grand Canyon, 2005

BACKGROUND Norovirus is often transmitted by infected food handlers at the point of service, whereas reports of food contamination before wholesale distribution are rare. In September 2005, we investigated reports of gastroenteritis among rafters who went on unrelated trips on the Colorado River. METHODS We surveyed all companies that launched rafting trips during the period from 14 August through 19 September 2005 to identify trips in which > or =3 rafters became ill. We conducted a case-control study. Case patients were persons who experienced diarrhea or vomiting that commenced < or =72 h after the trip launch; control subjects were persons who did not become ill < or =72 h after launch. We tested stool samples and food specimens for norovirus. We performed a traceback investigation of the suspected food vehicle and inspected the implicated processing plant. RESULTS Three or more rafters developed gastroenteritis during 13 (14%) of 91 trips, for a total of 137 ill persons. Of the 57 case patients who became ill < or =72 h after trip launch, 55 (96%) reported eating delicatessen meat, compared with 75 (79%) of 95 control subjects (odds ratio, 7.3; 95% confidence interval, 1.7-66.7). All delicatessen meat eaten by case patients came from 1 batch purchased from 1 processing plant and had been sliced, vacuum-packed, and frozen (temperature, -23 degrees C) for 7-28 days. An employee sliced this batch with bare hands 1 day after recovery from gastroenteritis. Identical norovirus sequences were identified in stool specimens obtained from rafters on 3 different trips; 2 of 5 meat packages also tested positive for norovirus by reverse-transcriptase polymerase chain reaction and DNA hybridization. CONCLUSIONS Food handlers can contaminate ready-to-eat meats with norovirus during processing. Meat-processing practices should include specific measures to prevent contamination with enteric viruses and subsequent widespread outbreaks.

Selection, Characterization and Application of Nucleic Acid Aptamers for the Capture and Detection of Human Norovirus Strains

Human noroviruses (HuNoV) are the leading cause of acute viral gastroenteritis and an important cause of foodborne disease. Despite their public health significance, routine detection of HuNoV in community settings, or food and environmental samples, is limited, and there is a need to develop alternative HuNoV diagnostic reagents to complement existing ones. The purpose of this study was to select and characterize single-stranded (ss)DNA aptamers with binding affinity to HuNoV. The utility of these aptamers was demonstrated in their use for capture and detection of HuNoV in outbreak-derived fecal samples and a representative food matrix. SELEX (Systematic Evolution of Ligands by EXponential enrichment) was used to isolate ssDNA aptamer sequences with broad reactivity to the prototype GII.2 HuNoV strain, Snow Mountain Virus (SMV). Four aptamer candidates (designated 19, 21, 25 and 26) were identified and screened for binding affinity to 14 different virus-like particles (VLPs) corresponding to various GI and GII HuNoV strains using an Enzyme-Linked Aptamer Sorbant Assay (ELASA). Collectively, aptamers 21 and 25 showed affinity to 13 of the 14 VLPs tested, with strongest binding to GII.2 (SMV) and GII.4 VLPs. Aptamer 25 was chosen for further study. Its binding affinity to SMV-VLPs was equivalent to that of a commercial antibody within a range of 1 to 5 µg/ml. Aptamer 25 also showed binding to representative HuNoV strains present in stool specimens obtained from naturally infected individuals. Lastly, an aptamer magnetic capture (AMC) method using aptamer 25 coupled with RT-qPCR was developed for recovery and detection of HuNoV in artificially contaminated lettuce. The capture efficiency of the AMC was 2.5–36% with an assay detection limit of 10 RNA copies per lettuce sample. These ssDNA aptamer candidates show promise as broadly reactive reagents for use in HuNoV capture and detection assays in various sample types.

Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII.4 strain

Human noroviruses (NoV) are the leading cause of acute viral gastroenteritis worldwide. Significant antigenic diversity of NoV strains has limited the availability of broadly reactive ligands for design of detection assays. The purpose of this work was to produce and characterize single stranded (ss)DNA aptamers with binding specificity to human NoV using an easily produced NoV target-the P domain protein. Aptamer selection was done using SELEX (Systematic Evolution of Ligands by EXponential enrichment) directed against an Escherichia coli-expressed and purified epidemic NoV GII.4 strain P domain. Two of six unique aptamers (designated M1 and M6-2) were chosen for characterization. Inclusivity testing using an enzyme-linked aptamer sorbent assay (ELASA) against a panel of 14 virus-like particles (VLPs) showed these aptamers had broad reactivity and exhibited strong binding to GI.7, GII.2, two GII.4 strains, and GII.7 VLPs. Aptamer M6-2 exhibited at least low to moderate binding to all VLPs tested. Aptamers significantly (p<0.05) bound virus in partially purified GII.4 New Orleans outbreak stool specimens as demonstrated by ELASA and aptamer magnetic capture (AMC) followed by RT-qPCR. This is the first demonstration of human NoV P domain protein as a functional target for the selection of nucleic acid aptamers that specifically bind and broadly recognize diverse human NoV strains.

An Enzyme-Linked Aptamer Sorbent Assay to Evaluate Aptamer Binding

Nucleic acid aptamers are a class of alternative ligands increasingly growing in importance in the face of contemporary detection challenges. Aptamers offer multiple advantages over traditional ligands like antibodies; however, their ability to specifically bind target molecules must first be confirmed after their generation. Use of a plate-based enzyme-linked aptamer sorbent assay (ELASA) is a generally rapid way to screen and characterize aptamer binding to protein targets. ELASA involves directly plating a protein target onto a nonspecific (polystyrene) surface and assessing binding of functionalized (biotinylated) aptamers to those plated proteins using an enzyme conjugate that recognizes the aptamers. Here, we describe an ELASA that was designed and used to evaluate and compare binding of ssDNA aptamers against the capsids of different strains of human norovirus.

Evaluation of a Surface Sampling Method for Recovery of Human Noroviruses Prior to Detection Using Reverse Transcription Quantitative PCR

Human noroviruses are the most common cause of acute viral gastroenteritis, and the environmental persistence of these viruses contributes to their transmissibility. Environmental sampling is thus an important tool for investigating norovirus outbreaks and for assessing the effectiveness of cleaning and decontamination regimens. The purpose of this study was to evaluate a sampling material (wipes) for their efficacy at recovering human norovirus from hard surfaces and foods. Dilutions of a human norovirus GII.4 stool specimen derived from an outbreak were applied to hard surfaces (stainless steel and ceramic) and the surfaces of representative foods (green pepper, apple, tomato, and cheese). The viruses were recovered at various times postinoculation using the wipes, followed by RNA extraction and reverse transcription quantitative PCR. Recovery efficiency ranged from 74% to almost 100% for all artificially inoculated hard surfaces and for most fresh produce surfaces. Less efficient recovery was observed for cheese. Viral RNA could be recovered from select surfaces for up to 7 days postinoculation, with a <1 log reduction in genome copy number. In field tests, 24 (11%) of 210 environmental samples collected during winter 2012 from restrooms in North Carolina were presumptively positive for human norovirus, and six of these samples were confirmed as GII.4 by sequencing. These wipes may be a valuable tool for investigations of norovirus outbreaks and studies of norovirus prevalence.