Gastroenteritis outbreaks associated with Norwalk-like viruses and their investigation by nested RT-PCR. (1/790)

BACKGROUND: Norwalk-like viruses are the most common cause of gastroenteritis outbreaks and sporadic cases of vomiting and diarrhoea. In healthy individuals infection is often mild and short-lived but in debilitated patients infection can be severe. It is essential that the virus laboratory can offer a sensitive and specific test, delivered in a timely manner. METHODS: We have developed a nested reverse transcriptase PCR based on published primers against the RNA polymerase gene and after comparison with electronmicroscopy used the assay to investigate 31 outbreaks of gastroenteritis. These were in diverse situations including nursing homes, small district hospitals, large general hospitals, a ferry ship, hotels, restaurants and staff canteens. RESULTS: A positive diagnosis was made in 30/31 outbreaks investigated giving an overall outbreak positive detection rate of 97%. At an individual patient level there was a positive diagnostic rate of 11.5% in a large hospital environment to 100% in smaller outbreak situations. The average patient positive rate was 34%. In addition we investigated 532 control faecal specimens from adults. Of these 530 were negative and 2 were repeatedly positive. CONCLUSIONS: It is essential that insensitive electronmicroscopy is replaced with the more sensitive reverse transcription PCR assays. These tests should be made available "on call" at weekends and public holidays. It is also important that outbreaks of NLV infection are monitored using sensitive RT-PCR assays so that the laboratory information can be used in ascertaining the spread and duration of the outbreak.  (+info)

Rapid and efficient extraction method for reverse transcription-PCR detection of hepatitis A and Norwalk-like viruses in shellfish. (2/790)

As part of an effort to develop a broadly applicable test for Norwalk-like viruses and hepatitis A virus (HAV) in shellfish, a rapid extraction method that is suitable for use with one-step reverse transcription (RT)-PCR-based detection methods was developed. The method involves virus extraction using a pH 9.5 glycine buffer, polyethylene glycol (PEG) precipitation, Tri-reagent, and purification of viral poly(A) RNA by using magnetic poly(dT) beads. This glycine-PEG-Tri-reagent-poly(dT) method can be performed in less than 8 h on hard-shell clams (Mercenaria mercenaria) and Eastern oysters (Crassostrea virginica) and, when coupled with RT-PCR-based detection, can yield results within 24 h. Observed sensitivities for seeded shellfish extracts are as low as 0.015 PFU of HAV and 22.4 RT-PCR50 units for Norwalk virus. Detection of HAV in live oysters experimentally exposed to contaminated seawater is also demonstrated. An adaptation of this method was used to identify HAV in imported clams (tentatively identified as Ruditapes philippinarum) implicated in an outbreak of food-borne viral illness. All of the required reagents are commercially available. This method should facilitate the implementation of RT-PCR testing of commercial shellfish.  (+info)

Characterization of capsid genes, expressed in the baculovirus system, of three new genetically distinct strains of "Norwalk-like viruses". (3/790)

"Norwalk-like viruses" (NLVs), members of a newly defined genus of the family Caliciviridae, are the most common agents of outbreaks of gastroenteritis in the United States. Two features of NLVs have hindered the development of simple methods for detection and determination of serotype: their genetic diversity and their inability to grow in cell culture. To assess the immune responses of patients involved in outbreaks of gastroenteritis resulting from infection with NLVs, we previously used recombinant-expressed capsid antigens representing four different genetic clusters, but this panel proved insufficient for detection of an immune response in many patients. To extend and further refine this panel, we expressed in baculovirus the capsid genes of three additional genetically distinct viruses, Burwash Landing virus (BLV), White River virus (WRV), and Florida virus. All three expressed proteins assembled into virus-like particles (VLPs) that contained a full-length 64-kDa protein, but both the BLV and WRV VLPs also contained a 58-kDa protein that resulted from deletion of 39 amino acids at the amino terminus. The purified VLPs were used to measure the immune responses in 403 patients involved in 37 outbreaks of acute gastroenteritis. A majority of patients demonstrated a fourfold rise in the titer of immunoglobulin G to the antigen homologous to the outbreak strain, but most seroconverted in response to other genetically distinct antigens as well, suggesting no clear pattern of type-specific immune response. Further study of the antigenicity of the NLVs by use of VLPs should allow us to design new detection systems with either broader reactivity or better specificity and to define the optimum panel of antigens required for routine screening of patient sera.  (+info)

Systemic, mucosal, and heterotypic immune induction in mice inoculated with Venezuelan equine encephalitis replicons expressing Norwalk virus-like particles. (4/790)

Norwalk-like viruses (NLVs) are a diverse group of single-stranded, nonenveloped, positive-polarity RNA viruses and are the leading cause of epidemic acute gastroenteritis in the United States. In this study, the major capsid gene of Norwalk virus, the prototype NLV, has been cloned and expressed in mammalian cells using a Venezuelan equine encephalitis (VEE) replicon expression system. Upon infection of baby hamster kidney (BHK) cells with VEE replicon particles (VRPs), the Norwalk virus capsid proteins self-assemble to generate high titers of Norwalk virus-like particles (VLPs) that are morphologically and antigenically analogous to wild-type Norwalk virus. Mice inoculated subcutaneously with VRPs expressing the Norwalk virus capsid protein (VRP-NV1) developed systemic and mucosal immune responses to Norwalk VLPs, as well as heterotypic antibody responses to the major capsid protein from another genogroup I NLV strain (NCFL) isolated from a recent outbreak. A second Norwalk virus capsid clone (NV2) containing three amino acid codon mutations from the NV1 clone was also expressed using VEE replicons (VRP-NV2), but upon infection of BHK cells failed to confer VLP self-assembly. Mice inoculated with VRP-NV2 elicited reduced systemic and mucosal immune responses to Norwalk VLPs, demonstrating the importance and potential utility of endogenous VLP presentation for maximum immune induction. Inoculation with either VRP-NV1 or VRP-NV2 resulted in serum antibody responses far superior to the induction in mice dosed orally with VLPs that were prepared using the VEE-NV1 replicon construct, a regimen similar to current models for NLV vaccination. Expression of NLV VLPs in mammalian cells offers a powerful approach for the design of novel NLV vaccines, either alone or in combination with current vaccination models.  (+info)

A predominant role for Norwalk-like viruses as agents of epidemic gastroenteritis in Maryland nursing homes for the elderly. (5/790)

Stool specimens from 156 Maryland nursing home residents, who became ill during 20 outbreaks of gastroenteritis from November 1987 through February 1988, were analyzed. All tested negative for astroviruses, enteroviruses, Group A rotaviruses, Sapporo-like caliciviruses, and enteric bacteria (i.e., Salmonella, Clostridium, and Shigella species). Eighty-two (52%) were positive for Norwalk-like viruses (NLVs), members of the family Caliciviridae. Six distinct genetic clusters within genogroups I and II of the NLVs were detected; a genogroup II (GII) virus closely related to the Camberwell virus in the NLV GII/4 genetic cluster was the predominant strain. Serologic evidence of infection with > or = 1 NLV was detected in 61 (56%) of 109 patients tested against 3 NLV antigens (i.e., Norwalk, Hawaii, and Toronto viruses). Sixteen (80%) outbreaks met the definition for an NLV outbreak. Taken together with a retrospective analysis of bacterial gastroenteritis in this same setting, these data support a major role for NLVs as etiologic agents of gastroenteritis in elderly persons.  (+info)

Identification of active-site amino acid residues in the Chiba virus 3C-like protease. (6/790)

The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.  (+info)

Waterborne outbreak of Norwalk-like virus gastroenteritis at a tourist resort, Italy. (7/790)

In July 2000, an outbreak of gastroenteritis occurred at a tourist resort in the Gulf of Taranto in southern Italy. Illness in 344 people, 69 of whom were staff members, met the case definition. Norwalk-like virus (NLV) was found in 22 of 28 stool specimens tested. The source of illness was likely contaminated drinking water, as environmental inspection identified a breakdown in the resort water system and tap water samples were contaminated with fecal bacteria. Attack rates were increased (51.4%) in staff members involved in water sports. Relative risks were significant only for exposure to beach showers and consuming drinks with ice. Although Italy has no surveillance system for nonbacterial gastroenteritis, no outbreak caused by NLV has been described previously in the country.  (+info)

Outbreak of acute gastroenteritis associated with Norwalk-like viruses among British military personnel--Afghanistan, May 2002. (8/790)

In the United States, Norwalk-like viruses (NLVs) cause an estimated 23 million episodes of illness, 50,000 hospitalizations, and 300 deaths each year. NLVs can be transmitted by fecally contaminated food and water and by direct person-to-person contact or through droplets of infected persons. Outbreaks of NLV-associated gastrointestinal illness are common in military settings. During May 13-19, 2002, a total of 29 British soldiers and staff of a field hospital in Afghanistan became acutely ill after a short incubation period with vomiting, diarrhea, and fever. This report summarizes the investigation of this outbreak and underscores the importance of the diagnostic capacity for NLVs.  (+info)