Characterization of "Norwalk-like viruses" and astroviruses by liquid hybridization assay.

"Norwalk-like viruses" (NLVs) and human astroviruses are causative agents of gastroenteritis in all age-groups. The typing of these agents is generally done by nucleotide sequencing, blot hybridization, or enzyme immunoassay. These techniques are expensive, time-consuming, and sometimes require scarce reagents, which limits the typing of NLVs and astroviruses to a few reference laboratories. This report describes a liquid hybridization assay that uses broadly reactive probes whose sequences are based on data from specimens in collections available at CDC and GenBank. Two astrovirus genogroup-specific probes were designed and tested successfully on 26 wild strains from all serotypes. Fourteen GII and 16 GI representative NLV strains were typed without cross-hybridization by using P1B- and P2A-specific probes, described previously, and new P2B- and P1A-specific probes. Analysis of the specificity of the probes, the effect of the mismatches during hybridization, and the sensitivity of hybridization assay demonstrates this method to be a rapid and simple technique for molecular typing of NLVs and preliminary characterization of astroviruses.

Genetic classification of "Norwalk-like viruses..

Reverse transcription-polymerase chain reaction has been used worldwide for the diagnosis of Norwalk-like virus (NLV) infection, yet a commonly accepted genetic classification scheme has not been established. Amino acid sequences from four regions of open-reading frame 2 (ORF2) were used to analyze 101 NLV strains, including 2 bovine strains. On the basis of this analysis, a genetic classification scheme is proposed that differentiates 99 human strains into 2 major genetic groups consisting of 5 and 10 genetic clusters, respectively. The 2 bovine strains constitute a newly defined third major genetic group composed of 2 putative clusters represented by each strain. This classification scheme is well supported by the analysis of the entire ORF2 sequences from 38 strains selected to represent the genetic diversity of the human strains used above. This scheme should provide a firm scientific basis for the unified classification of NLV strains detected around the world.

Identification of a distinct common strain of "Norwalk-like viruses" having a global distribution.

"Norwalk-like viruses" (NLVs) are the most common cause of outbreaks of nonbacterial gastroenteritis. During molecular surveillance of NLV strains from 152 outbreaks of gastroenteritis that occurred in the US between August 1993 and July 1997, we identified an NLV strain that predominated during the 1995-1996 season. The "95/96-US" strain caused 60 outbreaks in geographically distant locations within the US and was identified, by sequence comparisons, in an additional 7 countries on 5 continents during the same period. This is the first demonstration linking a single NLV strain globally and suggests that the circulation of these strains might involve patterns of transmission not previously considered. The diagnostic techniques are now available to establish a global network for surveillance of NLV strains that would highlight the importance of NLVs worldwide and allow molecular identification of common strains having a global distribution so as to consider interventions for their control.

Molecular epidemiology of "Norwalk-like viruses" in outbreaks of gastroenteritis in the United States.

Fecal specimens from 90 outbreaks of nonbacterial gastroenteritis reported to 33 state health departments from January 1996 to June 1997 were examined to determine the importance of and to characterize "Norwalk-like viruses" (NLVs) in these outbreaks. NLVs were detected by reverse transcription-polymerase chain reaction in specimens from 86 (96%) of 90 outbreaks. Outbreaks were most frequent in nursing homes and hospitals (43%), followed by restaurants or events with catered meals (26%); consumption of contaminated food was the most commonly identified mode of transmission (37%). Nucleotide sequence analysis showed great diversity between strains but also provided evidence indicating the emergence of a common, predominant strain. The application of improved molecular techniques to detect NLVs demonstrates that most outbreaks of nonbacterial gastroenteritis in the United States appear to be associated with these viruses and that sequence analysis is a robust tool to help link or differentiate these outbreaks.

Detection of norwalk-like virus in shellfish implicated in illness.

In the 1990s, Norwalk-like viruses (NLVs) were identified in patient specimens as the primary pathogen associated with shellfish-borne gastroenteritis in the United States. Identification of these viruses from implicated shellfish has been difficult due to inefficient recovery of viruses, natural polymerase chain reaction (PCR) inhibitors in shellfish, and low virus contamination. Recent improvements to the method of detecting NLVs in shellfish include enhanced processing of virus and shellfish samples, application of nested PCR and nucleotide sequencing, and increased knowledge of NLV genetic diversity. Using a newly developed and sensitive method, an NLV G2 strain was identified in 2 oyster samples implicated in a 1998 California outbreak involving 171 cases. NLV capsid primers demonstrated a greater specificity of PCR detection than did polymerase primers. The 175-base viral capsid nucleotide sequences derived from oysters were 100% identical to those derived from a patient stool sample. This finding supports the epidemiologic associations indicating that contaminated shellfish serve as the vehicle for NLV transmission.

Outbreak of Norwalk virus in a Caribbean island resort: application of molecular diagnostics to ascertain the vehicle of infection.

In 1998, an outbreak of gastroenteritis affected at least 448 persons including 122 staff at a resort hotel in Bermuda. A survey among staff indicated that gastroenteritis was associated with eating or drinking at the hotel (OR = 60, 95% CI = 2.4-15.1). Multiple specimens of drinking water had elevated faecal coliform levels and Escherichia coli present, suggestive of faecal contamination. Stools from 18 of the 19 persons with gastroenteritis that were tested were positive for genogroup-II Norwalk-like viruses (NLVs). RT-PCR analysis of a 31 specimen of water produced a genogroup-II NLV genome with a sequence identical to that of NLVs in the stools of three ill persons. This outbreak shows the value of new molecular diagnostics to link illness with a contaminated source through the use of sequence analysis. The risk of outbreaks such as these could be reduced in tourism dependent regions like Bermuda and the Caribbean by regular evaluation of data from the inspection and monitoring of drinking water supplies and waste water systems, by ensuring the chlorination of supplemental drinking water supplies and by establishing food-safety initiatives.

Development of methods to detect "Norwalk-like viruses" (NLVs) and hepatitis A virus in delicatessen foods: application to a food-borne NLV outbreak.

"Norwalk-like viruses" (NLVs) and hepatitis A virus (HAV) are the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for the detection of NLVs and HAV in foodstuffs. Although reverse transcription (RT)-PCR methods have been useful in detecting NLVs and HAV in bivalve mollusks implicated in outbreaks, to date such methods have not been available for other foods. To address this need, we developed a method to detect NLVs and HAV recovered from food samples. The method involves washing of food samples with a guanidinium-phenol-based reagent, extraction with chloroform, and precipitation in isopropanol. Recovered viral RNA is amplified with HAV- or NLV-specific primers in RT-PCRs, using a viral RNA internal standard control to identify potential sample inhibition. By this method, 10 to 100 PCR units (estimated to be equivalent to 10(2) to 10(3) viral genome copies) of HAV and Norwalk virus seeded onto ham, turkey, and roast beef were detected. The method was applied to food samples implicated in an NLV-associated outbreak at a university cafeteria. Sliced deli ham was positive for a genogroup II NLV as determined by using both polymerase- and capsid-specific primers and probes. Sequence analysis of the PCR-amplified capsid region of the genome indicated that the sequence was identical to the sequence from virus detected in the stools of ill students. The developed method is rapid, simple, and efficient.

Multistate outbreak of Norwalk-like virus gastroenteritis associated with a common caterer.

In February 2000, an outbreak of gastroenteritis occurred among employees of a car dealership in New York. The same meal was also supplied to 52 dealerships nationwide, and 13 states reported illness at dealerships where the banquet was served. A retrospective cohort study was conducted to identify risk factors associated with the illness. Stool samples were collected to detect Norwalk-like virus, and sera were drawn and tested for immunoglobulin A antibodies to the outbreak strain. By univariate analysis, illness was significantly associated with consumption of any of four salads served at the banquet (relative risk = 3.8, 95% confidence interval: 2.5, 5.6). Norwalk-like virus was detected by reverse transcription-polymerase chain reaction assay in 32 of 59 stool samples from eight states. Nucleotide sequences of a 213-base pair fragment from 16 stool specimens collected from cases in eight states were identical, confirming a common source outbreak. Two of 15 workers at caterer A had elevated immunoglobulin A titers to an antigenically related Norwalk-like virus strain. This study highlights the value of molecular techniques to complement classic epidemiologic methods in outbreak investigations and underscores the critical role of food handlers in the spread of foodborne disease associated with Norwalk-like virus.

A foodborne outbreak of gastroenteritis associated with Norwalk-like viruses: first molecular traceback to deli sandwiches contaminated during preparation.

In March 1998, an outbreak of acute gastroenteritis occurred among students at a Texas university. Overall, 125 ill students sought medical care. Case-control studies revealed that illness was significantly associated with eating foods from the university's main cafeteria deli bar on 9 and 10 March. Stool specimens from 9 (50%) of 18 ill students and samples of deli ham showed evidence of Norwalk-like viruses (NLVs) by reverse-transcriptase (RT) polymerase chain reaction (PCR) assay. A food handler who prepared sandwiches for lunch on 9 March reported that her infant had been sick with watery diarrhea since just before the outbreak. A stool sample from the infant was positive for NLV by RT-PCR, and the sequence of the amplified product was identical to that of amplified product from deli ham and students' stool specimens. This is the first time RT-PCR and sequence analysis have successfully confirmed viral contamination of a food item likely to have been contaminated by a food handler.

"Norwalk-like viruses". Public health consequences and outbreak management.

"Norwalk-like viruses" (NLVs) cause outbreaks of gastroenteritis and are spread frequently through contaminated food or water. Molecular diagnostics now enables detecting viruses in clinical and environmental specimens, linking of NLV strains causing outbreaks in multiple geographic locations, and tracing them to their sources in contaminated food or water. This report reviews recent advances in NLV detection and provides guidelines and recommendations for investigating NLV-related outbreaks, including specimen collection and disease prevention and control. This report also updates information provided in CDC's previously published, Viral Agents of Gastroenteritis: Public Health Importance and Outbreak Management (MMWR 1990;39 [No. RR-5]: 1-24). These CDC recommendations are intended for public health professionals who investigate outbreaks of acute gastroenteritis but could be useful in academic and research settings as well.

An outbreak of viral gastroenteritis associated with consumption of sandwiches: implications for the control of transmission by food handlers.

Although food handlers are often implicated as the source of infection in outbreaks of food-borne viral gastroenteritis, little is known about the timing of infectivity in relation to illness. We investigated a gastroenteritis outbreak among employees of a manufacturing company and found an association (RR = 14.1, 95% CI = 2.0-97.3) between disease and eating sandwiches prepared by 6 food handlers, 1 of whom reported gastroenteritis which had subsided 4 days earlier. Norwalk-like viruses were detected by electron microscopy or reverse transcriptase-polymerase chain reaction (RT-PCR) in stool specimens from several company employees, the sick food handler whose specimen was obtained 10 days after resolution of illness, and an asymptomatic food handler. All RT-PCR product sequences were identical, suggesting a common source of infection. These data support observations from recent volunteer studies that current recommendations to exclude food handlers from work for 48-72 h after recovery from illness may not always prevent transmission of Norwalk-like viruses because virus can be shed up to 10 days after illness or while exhibiting no symptoms.

Norwalk-like viral gastroenteritis outbreak in U.S. Army trainees.

An outbreak of acute gastroenteritis hospitalized 99 (12%) of 835 U. S. Army trainees at Fort Bliss, El Paso, Texas, from August 27 to September 1, 1998. Reverse transcriptase polymerase chain reaction tests for Norwalk-like virus were positive for genogroup 2. Gastroenteritis was associated with one post dining facility and with soft drinks.