RESUMEN

Noroviruses (NoV) are a leading cause of epidemic gastroenteritis. Human challenge studies have been used to examine the infectivity, pathogenicity, and host immune response to NoV as well as vaccine efficacy. The goal of this study was to conduct a meta-analysis of data from five previously completed human challenge trials and compare the response to the secondary NV inoculum (8fIIb) to its precursor (8fIIa). We investigated a total of 158 subjects: 76 subjects were experimentally challenged with NV inoculum 8fIIa, and 82 subjects were challenged with 8fIIb. We compared demographic characteristics, infection, illness, mean severity score, blood types, and duration of viral shedding between the two groups of subjects. There were no statistically significant differences in overall infection and illness rates between subjects inoculated with 8fIIa and 8fIIb. However, individuals challenged with 8fIIa had significantly higher severity scores (5.05 vs. 3.22, p = .008) compared with those challenged with 8fIIb. We also observed that infection with 8fIIb was associated with significantly longer duration of viral shedding compared with 8fIIa (11.0 days vs. 5.0 days, p = .0005). These results have serious implications for the development of new NoV inocula for human challenge studies to test candidate vaccine efficacy-where illness severity and duration of viral shedding are important outcomes.

Asunto(s)

Infecciones por Caliciviridae/virología , Virus Norwalk/clasificación , Virus Norwalk/patogenicidad , Esparcimiento de Virus , Adolescente , Adulto , Infecciones por Caliciviridae/inmunología , Relación Dosis-Respuesta Inmunológica , Femenino , Gastroenteritis/virología , Voluntarios Sanos , Experimentación Humana/estadística & datos numéricos , Humanos , Masculino , Persona de Mediana Edad , Virus Norwalk/genética , Virus Norwalk/inmunología , Índice de Severidad de la Enfermedad , Adulto Joven

RESUMEN

BACKGROUND: Human noroviruses (HuNoV) are the leading cause of gastroenteritis. No vaccine is currently available to prevent norovirus illness or infection. Safe, infectious challenge strains are needed to assess vaccine efficacy in the controlled human infection model (CHIM). METHODS: A stock of HuNoV strain Norwalk virus ([NV] GI.1) was prepared. Healthy, genetically susceptible adults were inoculated with NV Lot 001-09NV and monitored for infection, gastroenteritis symptoms, and immune responses. RESULTS: Lot 001-09NV induced gastroenteritis in 9 (56%) and infection in 11 (69%) of 16 genetically susceptible subjects. All infected subjects developed strong immune responses to GI.1 with a 30-fold (geometric mean titer) increase in blocking titers (BT50) and a 161-fold increase in GI.1-specific immunoglobulin (Ig)G titers when compared with baseline. GI.1-specific cellular responses in peripheral blood were observed 9 days postchallenge with an average of 3253 IgA and 1227 IgG antibody-secreting cells per million peripheral blood mononuclear cells. CONCLUSIONS: GI.1 Lot 001-09NV appears to be similar in virulence to previous passages of NV strain 8fIIa. The safety profile, attack rate, and duration of illness make GI.1 Lot 001-09NV a useful challenge strain for future vaccine studies aimed at establishing immune correlates.

Asunto(s)

Infecciones por Caliciviridae/prevención & control , Infecciones por Caliciviridae/virología , Gastroenteritis/prevención & control , Gastroenteritis/virología , Virus Norwalk/clasificación , Vacunas Virales/inmunología , Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto Joven

RESUMEN

The human noroviruses (NoVs) are genetically diverse, rapidly evolving RNA viruses and are the major cause of epidemic gastroenteritis of humans. Serum antibodies that block the interaction of NoVs and NoV viruslike particles (VLPs) with host attachment factors are considered surrogate neutralizing antibodies in the absence of cell culture and small-animal replication models for the human NoVs. A serological assay for NoV-blocking antibodies was used to assess the breadth of the heterotypic antibody response in the context of an experimental challenge study with a human NoV. Heterotypic histo-blood group antigen (HBGA)-blocking activity against GI.4, GI.7, and GII.4 NoVs increased significantly in the serum of individuals (n = 18) infected with Norwalk virus (GI.1). Although the fold increases and peak titers of heterotypic antibody were more modest than titers of antibody reactive with the challenge antigen, Norwalk virus infection elicited a serological rise even against the novel Sydney variant of GII.4 NoVs. These observations indicate that the development of a broadly cross-protective NoV vaccine containing a limited number of genotypes may be possible.

Asunto(s)

Anticuerpos Neutralizantes/sangre , Anticuerpos Antivirales/sangre , Infecciones por Caliciviridae/inmunología , Reacciones Cruzadas , Virus Norwalk/inmunología , Adulto , Infecciones por Caliciviridae/virología , Estudios de Cohortes , Genotipo , Humanos , Virus Norwalk/clasificación , Virus Norwalk/genética

RESUMEN

Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI-GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer substrates. We demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases was inhibited by commercial protease inhibitors including chymostatin. The interaction of chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy.

Asunto(s)

Gastroenteritis/virología , Virus Norwalk/enzimología , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Regulación hacia Abajo , Transferencia Resonante de Energía de Fluorescencia , Genotipo , Humanos , Cinética , Datos de Secuencia Molecular , Norovirus/química , Norovirus/clasificación , Norovirus/enzimología , Norovirus/genética , Virus Norwalk/química , Virus Norwalk/clasificación , Virus Norwalk/genética , Oligopéptidos/química , Oligopéptidos/farmacología , Péptido Hidrolasas/genética , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Alineación de Secuencia , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/genética

RESUMEN

Human noroviruses are difficult to study due to the lack of an efficient in vitro cell culture system or small animal model. Murine norovirus replicates in murine macrophages (MPhi) and dendritic cells (DCs), raising the possibility that human NoVs might replicate in such human cell types. To test this hypothesis, we evaluated DCs and MPhi derived from monocyte subsets and CD11c(+) DCs isolated from peripheral blood mononuclear cells of individuals susceptible to Norwalk virus (NV) infection. These cells were exposed to NV and replication was evaluated by immunofluorescence and by quantitative RT-PCR. A few PBMC-derived DCs expressed NV proteins. However, NV RNA did not increase in any of the cells tested. These results demonstrate that NV does not replicate in human CD11c(+) DCs, monocyte-derived DCs and MPhi, but abortive infection may occur in a few DCs. These results suggest that NV tropism is distinct from that of murine noroviruses.

Asunto(s)

Células Dendríticas/virología , Macrófagos/virología , Virus Norwalk/fisiología , Sistema del Grupo Sanguíneo ABO , Adulto , Animales , Antígenos Virales/metabolismo , Secuencia de Bases , Receptor 1 de Quimiocinas CX3C , Infecciones por Caliciviridae/genética , Infecciones por Caliciviridae/fisiopatología , Infecciones por Caliciviridae/virología , Cartilla de ADN/genética , Células Dendríticas/clasificación , Células Dendríticas/inmunología , Fucosiltransferasas/genética , Proteínas Ligadas a GPI , Genotipo , Humanos , Técnicas In Vitro , Interferón Tipo I/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Macrófagos/inmunología , Ratones , Virus Norwalk/clasificación , Virus Norwalk/patogenicidad , Receptores de Quimiocina/metabolismo , Receptores de IgG/metabolismo , Especificidad de la Especie , Tropismo Viral , Replicación Viral , Galactósido 2-alfa-L-Fucosiltransferasa

RESUMEN

Norovirus immunity is poorly understood as the limited data available on protection after infection are often contradictory. In contrast to the more prominent GII noroviruses, GI norovirus infections are less frequent in outbreaks. The GI noroviruses display very complex patterns of heterotypic immune responses following infection, and many individuals are highly susceptible to reinfection. To study the immune responses and mechanisms of GI.1 persistence, we built structural models and recombinant virus-like particles (VLPs) of five GI strains: GI.1-1968, GI.1-2001, GI.2-1999, GI.3-1999, and GI.4-2000. Structural models of four GI genotype capsid P domain dimers suggested that intragenotype structural variation is limited, that the GI binding pocket is mostly preserved between genotypes, and that a conserved, surface-exposed epitope may allow for highly cross-reactive immune responses. GI VLPs bound to histo-blood group antigens (HBGAs) including fucose, Lewis, and A antigens. Volunteers infected with GI.1-1968 (n = 10) had significant increases between prechallenge and convalescent reactive IgG for all five GI VLPs measured by enzyme immunoassay. Potential cross-neutralization of GI VLPs was demonstrated by convalescent-phase serum cross-blockade of GI VLP-HBGA interaction. Although group responses were significant for all GI VLPs, each individual volunteer demonstrated a unique VLP blockade pattern. Further, peripheral blood mononuclear cells (PBMCs) were stimulated with each of the VLPs, and secretion of gamma interferon (IFN-gamma) was measured. As seen with blockade responses, IFN-gamma secretion responses differed by individual. Sixty percent responded to at least one GI VLP, with only two volunteers responding to GI.1 VLP. Importantly, four of five individuals with sufficient PBMCs for cross-reactivity studies responded more robustly to other GI VLPs. These data suggest that preexposure history and deceptive imprinting may complicate PBMC and B-cell immune responses in some GI.1-1968-challenged individuals and highlight a potential complication in the design of efficacious norovirus vaccines.

Asunto(s)

Infecciones por Caliciviridae/inmunología , Infecciones por Caliciviridae/virología , Gastroenteritis/inmunología , Gastroenteritis/virología , Virus Norwalk/inmunología , Secuencia de Aminoácidos , Anticuerpos Antivirales/sangre , Antígenos Virales/química , Antígenos Virales/genética , Reacciones Cruzadas , Genotipo , Humanos , Inmunidad Celular , Inmunidad Humoral , Inmunoglobulina G/sangre , Interferón gamma/sangre , Modelos Moleculares , Datos de Secuencia Molecular , Virus Norwalk/clasificación , Virus Norwalk/genética , Virus Norwalk/patogenicidad , Filogenia , Homología de Secuencia de Aminoácido , Linfocitos T/inmunología , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/inmunología , Virión/inmunología

RESUMEN

Tracking the spread of noroviruses during outbreaks of gastroenteritis is hampered by the lack of sequence diversity in those regions of the genome chosen for virus detection and characterization. Sequence analysis of regions of the genes encoding the RNA-dependent RNA polymerase and the S domain of the capsid does not provide sufficient discrimination between genotypically related strains of different outbreaks. However, analysis of sequences derived from the region encoding the P2 domain showed 100% similarity among strains from the same outbreak and <100% similarity among strains of different outbreaks. The prolonged nature of some hospital outbreaks, links between hospitals, and the introduction of multiple strains of a single genotype associated with an outbreak aboard a cruise ship were determined using this method. This provides a powerful tool for tracking outbreak strains and the subsequent analysis and validation of interventions in a background of multiple introductions of virus strains of the same genotype or genetic cluster.

Asunto(s)

Infecciones por Caliciviridae , Proteínas de la Cápside/genética , Brotes de Enfermedades , Gastroenteritis , Virus Norwalk/clasificación , Virus Norwalk/genética , Análisis de Secuencia de ADN , Secuencia de Bases , Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/transmisión , Infecciones por Caliciviridae/virología , Heces/virología , Gastroenteritis/epidemiología , Gastroenteritis/virología , Genotipo , Hospitales , Humanos , Virus Norwalk/química , Virus Norwalk/aislamiento & purificación , Filogenia , Reacción en Cadena de la Polimerasa , ARN Viral/análisis , ARN Viral/aislamiento & purificación , Navíos , Especificidad de la Especie , Reino Unido/epidemiología

RESUMEN

Between July 2000 and June 2004, fecal specimens from 270 outbreaks of acute gastroenteritis were sent to the Centers for Disease Control and Prevention by local or state health departments for calicivirus testing. Of the 226 outbreaks that met the criteria for inclusion in the present study, caliciviruses were detected in 184 (81%) by reverse-transcription polymerase chain reaction and nucleotide sequencing. Nursing homes, retirement centers, and hospitals were the most frequently reported settings, and person-to-person contact was the most common mode of transmission, followed by foodborne spread. Overall, genogroup II norovirus (NoV) strains were the most abundant (79%), followed by genogroup I NoV strains (19%) and sapovirus (2%). Nucleotide-sequence analysis indicated a great diversity of NoV strains and implicated the emergence of one particular sequence variant in outbreaks occurring between July 2002 and June 2003. The public health impact of caliciviruses will not be fully appreciated, nor will interventions be completely evaluated, until methods to detect these viruses are more routinely used.

Asunto(s)

Infecciones por Caliciviridae/epidemiología , Caliciviridae/genética , Brotes de Enfermedades , Gastroenteritis/epidemiología , Epidemiología Molecular , Enfermedad Aguda , Caliciviridae/clasificación , Caliciviridae/aislamiento & purificación , Infecciones por Caliciviridae/virología , Gastroenteritis/virología , Humanos , Virus Norwalk/clasificación , Virus Norwalk/genética , Virus Norwalk/aislamiento & purificación , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estaciones del Año , Análisis de Secuencia de ADN , Estados Unidos/epidemiología

Asunto(s)

Infecciones por Caliciviridae , Gastroenteritis/virología , Virus Norwalk , Enfermedad Aguda , Diagnóstico Diferencial , Ensayo de Inmunoadsorción Enzimática , Gastroenteritis/diagnóstico , Gastroenteritis/prevención & control , Gastroenteritis/terapia , Genotipo , Humanos , Virus Norwalk/clasificación , Virus Norwalk/genética , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

Asunto(s)

Virus Norwalk , Infecciones por Caliciviridae/diagnóstico , Diagnóstico Diferencial , Gastroenteritis/virología , Genoma Viral , Humanos , Técnicas de Diagnóstico Molecular , Virus Norwalk/clasificación , Virus Norwalk/genética , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Viral pollution in shellfish has been analyzed simultaneously across a wide range of geographical regions, with emphasis on the concomitant variations in physicochemical characteristics and social features. The methods for sample treatment and for the detection of human enteric viruses were optimized by the participating laboratories. The second part of this study involves the selection of a protocol for virus detection, which was validated by analyzing the distribution and concentration of human viral pathogens under diverse conditions during an 18-month period in four European countries. Shellfish-growing areas from diverse countries in the north and south of Europe were defined and studied, and the microbiological quality of the shellfish was analyzed. Human adenovirus, Norwalk-like virus, and enterovirus were identified as contaminants of shellfish in all the participating countries. Hepatitis A virus was also isolated in all areas except Sweden. The seasonal distribution of viral contamination was also described. Norwalk-like virus appeared to be the only group of viruses that demonstrated seasonal variation, with lower concentrations occurring during warm months. The depuration treatments currently applied were shown to be adequate for reducing Escherichia coli levels but ineffective for the elimination of viral particles. The human adenoviruses detected by PCR correlate with the presence of other human viruses and could be useful as a molecular index of viral contamination in shellfish.

Asunto(s)

Adenovirus Humanos/aislamiento & purificación , Enterovirus/aislamiento & purificación , Virus de la Hepatitis A/aislamiento & purificación , Virus Norwalk/aislamiento & purificación , Mariscos/microbiología , Animales , Enterovirus/clasificación , Reacciones Falso Negativas , Grecia , Humanos , Virus Norwalk/clasificación , Filogenia , España , Suecia , Reino Unido

RESUMEN

Viruses of the genus 'Norwalk-like viruses' (NLVs) detected in humans have been genetically classified into two major genetic groups, genogroups I and II (GI and GII), which together are made up of at least 14 genetic subgroups. However, a comparable classification of NLVs in other species remains to be carried out. We sequenced a 2-kb region from within the RNA polymerase gene to the 3' end of open reading frame 2 (ORF2) of two NLV strains previously detected in the caecum contents of healthy pigs. The sequences of the entire ORF2 of these two NLV strains were analyzed for their genetic relationships to 15 human strains, which have already been reported and used as references for the genetic classification of human NLV strains, and additional two strains; one, a human strain which has recently been reported and appears to represent a new genetic subgroup of GII; and the other, an animal NLV strain. Analysis of a matrix showing pairwise identities and topology of a neighbor-joining tree showed that the two swine strains could be classified into a new genetic subgroup of GII on the basis of the amino acid sequences of the entire capsid protein. Grouping of the two swine strains was well corroborated by results of similar analyses of nucleotide sequences of the entire ORF2 and of a 510 base region at the 3' end of ORF1.

Asunto(s)

Portador Sano , Ciego/virología , Virus Norwalk/clasificación , Animales , Infecciones por Caliciviridae/virología , ARN Polimerasas Dirigidas por ADN , Genoma Viral , Datos de Secuencia Molecular , Virus Norwalk/genética , Sistemas de Lectura Abierta , Filogenia , Análisis de Secuencia de ADN , Porcinos

RESUMEN

Acute gastroenteritis due to Norwalk virus(NV), a member of the family Caliciviridae, is a common illness worldwide. NV is a human pathogen causing food-borne and water-borne diseases that occur in various epidemiological settings. The disease is mild and self-limiting and the symptomatic phase lasts 24 to 72 hours. Genetic analyses of the polymerase and the capsid protein indicate that NV is grouped into two clusters, genogroup I and genogroup II. The development of recombinant virus-like particles made a great impact on the development of diagnostic assays such as antigen ELISA and antibody ELISA. Strong control measures are needed to prevent oyster-related outbreaks.

Asunto(s)

Infecciones por Caliciviridae/virología , Gastroenteritis/virología , Virus Norwalk , Ensayo de Inmunoadsorción Enzimática , Microbiología de Alimentos , Genotipo , Humanos , Virus Norwalk/clasificación , Virus Norwalk/genética , Virus Norwalk/aislamiento & purificación , Reacción en Cadena de la Polimerasa

RESUMEN

In a recent study, RNA with nucleotide sequeces specific for "Norwalk-like viruses" (NLV) was detected in 11 different brands of European mineral waters. To clarify this finding, a 1-year monitoring study was conducted. Samples of three European brands of mineral water without gas were monitored weekly by reverse transcriptase PCR using generic and genogroup-specific oligonucleotides. Additional analyses were performed to investigate a possible correlation between NLV sequence contamination and mineral water lot numbers, the long-term stability (persistence) of NLV sequences in mineral water, and the level of contamination. NLV sequences were detected in 53 of 159 samples analyzed (33%) and belonged entirely to genogroup II. Although all NLV strains identified were closely related, three mineral water brand-specific clusters could be identified for both primer systems by sequencing. Analyses of second samples from lots previously shown to be positive for NLV sequences gave corresponding results in 45 of 53 cases (85%) (within a six-pack). NLV persistence was tested by analyzing 10 positive samples after 6 and 12 months of storage in darkness at room temperature. After 6 months, all samples remained positive; after 12 months, 9 of 10 samples were still positive for NLV sequences. No NLV sequences could be detected by analysis of 0.1-liter aliquots of 53 samples shown to be positive by testing of 1-liter volumes. Based on this fact and a test sensitivity of approximately 10 viral units, levels of contamination in positive mineral water samples were estimated to be in the range of 10 to 100 genomic equivalents per liter.

Asunto(s)

Aguas Minerales/virología , Virus Norwalk/clasificación , Virus Norwalk/aislamiento & purificación , Filtración/métodos , Datos de Secuencia Molecular , Virus Norwalk/genética , Filogenia , ARN Viral , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN

RESUMEN

Norwalk-like viruses (NLVs) are now established as the most important causative agents of epidemic gastroenteritis worldwide. The overall objective of this study was to determine the molecular epidemiology of Irish NLV isolates for the first time by obtaining sequence data from specimens originating from outbreaks and sporadic cases of gastroenteritis. Eight samples from sporadic cases of gastroenteritis and nine isolates from separate NLV outbreaks were examined. Of the sporadic isolates, six were shown to be genogroup 2 (G2) by RT-PCR, while two were G1. All of the outbreak isolates were G2. All isolates were partially sequenced within a highly conserved region of ORF1 (RNA-dependent RNA polymerase gene). Sequence data were aligned and a dendogram was constructed. The results indicated that the majority of G2 isolates were seen to cluster with Bristol and Lordsdale virus, while the two G1 specimens were related most closely to Southampton virus. Further downstream sequence analysis of a number of the isolates confirmed this result. It is concluded that the majority of NLV isolates circulating in Ireland belong to the Bristol/Lordsdale clade.

Asunto(s)

Infecciones por Caliciviridae/epidemiología , Brotes de Enfermedades , Gastroenteritis/epidemiología , Virus Norwalk/genética , Secuencia de Bases , Humanos , Irlanda/epidemiología , Epidemiología Molecular , Datos de Secuencia Molecular , Virus Norwalk/clasificación , Virus Norwalk/enzimología , Filogenia , ARN Viral/genética , ARN Polimerasa Dependiente del ARN/genética , Alineación de Secuencia

RESUMEN

A total of 177 different nucleotide sequences of the RNA polymerase region of Norwalk-like viruses (NLVs) genomes, collected via a nation-wide survey project in Japan between 1989 and 1998, were examined by reverse transcription-polymerase chain reaction (RT-PCR) employing various primer pairs. The nucleotide sequences of different strains showed great diversity, with a range of 57 to 100% identities among strains. The strains could be classified into five clusters: Norwalk (NV), Snow Mountain agent/Bristol virus (SMA/BV), Toronto virus/Mexico virus (TV/MX), and Japan specific cluster 1 and 2 (JP-1 and JP-2). Within each cluster there is greater than 85% identity of amino acid sequence (more than 75% identity of nucleotide sequences), based on sequence homology analysis. We believe that two of the five clusters, JP-1and JP-2, define new specific clusters found in Japan according to phylogenetic and pair-wise comparison studies. An RT-PCR procedure was designed using new consensus primer pairs, P1/P2, P1/P3, and Y1/Y2 based on multiple alignment of collected nucleotide sequences, that are expected to detect nearly all NLVs prevailing in Japan. The usefulness of the primers was tested by ten different laboratories in Japan using a panel of ten fecal samples containing different virus strains. The identification of these primer pairs will facilitate routine diagnosis of NLV infection by RT-PCR and offers the potential for their direct detection in food and environmental samples.

Asunto(s)

Infecciones por Caliciviridae/virología , Cartilla de ADN/genética , Brotes de Enfermedades , Virus Norwalk/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Caliciviridae/epidemiología , Variación Genética , Humanos , Japón/epidemiología , Epidemiología Molecular , Virus Norwalk/clasificación , Virus Norwalk/aislamiento & purificación , Filogenia , ARN Viral/genética , Análisis de Secuencia de ARN , Homología de Secuencia

RESUMEN

Reverse transcription-PCR and sequence analysis identified calciviruses in 32 of 60 stool specimens (negative for other enteric pathogens) obtained from children admitted to our hospital with acute gastroenteritis. The overall annual incidence rate for calcivirus was 9% (32 of 354 children). Molecular analysis identified 30 "Norwalk-like virus" genogroup II (predominantly Lordsdale cluster) and 2 "Sapporo-like virus" strains.

Asunto(s)

Infecciones por Caliciviridae/epidemiología , Caliciviridae/aislamiento & purificación , Gastroenteritis/epidemiología , Virus Norwalk/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedad Aguda , Australia/epidemiología , Caliciviridae/clasificación , Caliciviridae/genética , Infecciones por Caliciviridae/virología , Preescolar , Gastroenteritis/virología , Hospitalización , Humanos , Lactante , Virus Norwalk/clasificación , Virus Norwalk/genética , Filogenia , Análisis de Secuencia de ADN

RESUMEN

We report the case of an elderly woman excreting high levels (about 5 x 10(5) virions per gram of faeces) of Norwalk-like virus (NLV) in the absence of any clinical symptoms of gastroenteritis. Analysis by reverse transcription, polymerase chain reaction and DNA sequencing was carried out on a 342-nucleotide region of open reading frame 1. This indicated that the NLV belonged to genogroup 2 and was more closely related to the Camberwell subgroup, the most common circulating in southeast Australia at present, than to the Norwalk and Mexico viruses.

Asunto(s)

Infecciones por Caliciviridae/virología , Heces/virología , Gastroenteritis/virología , Virus Norwalk/aislamiento & purificación , Esparcimiento de Virus , Anciano , Cartilla de ADN/química , ADN Viral/análisis , Femenino , Humanos , Datos de Secuencia Molecular , Virus Norwalk/clasificación , Virus Norwalk/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN

RESUMEN

"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.

Asunto(s)

Mamastrovirus/clasificación , Virus Norwalk/clasificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones por Astroviridae/epidemiología , Infecciones por Astroviridae/virología , Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/virología , ADN Viral/metabolismo , Brotes de Enfermedades , Heces/virología , Gastroenteritis/epidemiología , Gastroenteritis/virología , Genes Virales , Genotipo , Humanos , Mamastrovirus/genética , Mamastrovirus/aislamiento & purificación , Virus Norwalk/genética , Virus Norwalk/aislamiento & purificación , Ácidos Nucleicos Heterodúplex , Sondas de Oligonucleótidos , Sistemas de Lectura Abierta/genética , Filogenia , ARN Viral/análisis , ARN Viral/genética , ARN Viral/aislamiento & purificación , Sensibilidad y Especificidad , Especificidad de la Especie

RESUMEN

Norwalk-like viruses (NLVs) is a genus belonging to the Caliciviridae. NLVs are transmitted by the fecal-oral and the aerosol route and are the most common cause of outbreaks of nonbacterial gastroenteritis. NLVs are responsible for an estimated 67% of all illnesses caused by known foodborne pathogens and for 96% of nonbacterial gastroenteritis in the United States. Many outbreaks could be associated with the consumption of primarily or secondarily contaminated foods. To our knowledge, no epidemic arising from contaminated mineral water has been reported. We investigated the presence of NLV sequences in 63 mineral waters of 29 different brands that were imported into or bottled in Switzerland. NLV sequences were detected in 21 mineral waters by reverse transcription-seminested polymerase chain reaction. Specimens of two NLV genogroups (gg), gg I and gg II, were randomly present in the contaminated samples. The presence of NLV sequences could not be correlated either with bottle characteristics or with chemical properties like mineralization, pH, or the presence of carbonic acid. Nucleotide sequence analysis of 12 NLV-positive samples revealed several point mutations. All isolated NLV gg I strains have a similarity of 70 to 87% with the common Desert Shield virus (UO4469), and all isolated NLV gg II strains have a similarity of 89 to 93% with the Camberwell virus (U46500). Possible reasons for the presence of NLV sequences in mineral waters are discussed.

Asunto(s)

Aguas Minerales/virología , Virus Norwalk/aislamiento & purificación , Microbiología del Agua , ADN Viral/análisis , ADN Viral/química , Brotes de Enfermedades , Datos de Secuencia Molecular , Virus Norwalk/clasificación , Virus Norwalk/genética , Mutación Puntual , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Suiza