A large outbreak of acute gastroenteritis associated with astrovirus among students and teachers in Osaka, Japan.

In June 1991, a large outbreak of acute nonbacterial gastroenteritis occurred among students and teachers at 10 primary and 4 junior high schools in Katano City, Osaka, Japan. The outbreak affected > 4700 persons, lasted 5 days, and was believed to have been linked to contaminated food from a common supplier. Astrovirus, identified as the etiologic agent, was detected by direct electron microscopy in 10 of 38 fecal samples obtained from patients with diarrhea. Detection was confirmed by solid-phase immune electron microscopy (IEM), EIA, reverse transcription-polymerase chain reaction, and virus isolation in CaCo-2 cells. Several patients who had astrovirus in their stool also demonstrated a significant antibody response to a reference strain of astrovirus by IEM and EIA and to their own isolate by IEM. Astrovirus can be an important agent of epidemic acute nonbacterial gastroenteritis in school-aged children and adults in Japan.

RNA sequence of astrovirus: distinctive genomic organization and a putative retrovirus-like ribosomal frameshifting signal that directs the viral replicase synthesis.

The genomic RNA of human astrovirus was sequenced and found to contain 6797 nt organized into three open reading frames (1a, 1b, and 2). A potential ribosomal frameshift site identified in the overlap region of open reading frames 1a and 1b consists of a "shifty" heptanucleotide and an RNA stem-loop structure that closely resemble those at the gag-pro junction of some retroviruses. This translation frame-shift may result in the suppression of in-frame amber termination at the end of open reading frame 1a and the synthesis of a nonstructural, fusion polyprotein that contains the putative protease and RNA-dependent RNA polymerase. Comparative sequence analysis indicated that the protease and polymerase of astrovirus are only distantly related to the respective enzymes of other positive-strand RNA viruses. The astrovirus polyprotein lacks the RNA helicase domain typical of other positive-strand RNA viruses of similar genome size. The genomic organization and expression strategy of astrovirus, with the protease and the polymerase brought together by predicted frameshift, most closely resembled those of plant leuteoviruses. Specific features of the sequence and genomic organization support the classification of astroviruses as an additional family of positive-strand RNA viruses, designated Astroviridae.

Detection of antibody to recombinant Norwalk virus antigen in specimens from outbreaks of gastroenteritis.

Norwalk virus and other small round-structured viruses are commonly associated with outbreaks of gastroenteritis. We used a recently described recombinant-expressed Norwalk virus (rNV) capsid protein in enzyme immunoassays to quantitatively measure immunoglobulin G (IgG) and IgA to Norwalk virus in serum pairs from patients involved in outbreaks of gastroenteritis. The outbreaks previously were classified, on the basis of the results of a blocking antibody assay, as Norwalk virus negative, serologically intermediate, or Norwalk virus positive. The rNV IgG assay was more sensitive than the blocking assay for detecting IgG to Norwalk virus in serum from patients in all outbreak classes. There was 79% concordance between seroconversions detected by the blocking antibody assay and those detected by the rNV IgG assay. The rNV IgA assay detected seroconversions to Norwalk virus primarily in patients involved in outbreaks previously classified as Norwalk virus positive.

Subgenomic RNA sequence of human astrovirus supports classification of Astroviridae as a new family of RNA viruses.

We report the sequence of the subgenomic RNA of human astrovirus serotype 2. This 2,484-nucleotide RNA contains a single open reading frame, which encodes a protein with a predicted molecular mass of 88 kDa. We propose that this protein is the 90-kDa capsid precursor observed in infected cells. The deduced protein sequence does not contain conserved amino acid patterns reported for the capsid proteins of picornaviruses or caliciviruses, consistent with the classification of astroviruses as a new family of RNA viruses, designated Astroviridae.

A multistate outbreak of Norwalk virus gastroenteritis associated with consumption of commercial ice.

Between 19 and 27 September 1987, a cluster of outbreaks of gastrointestinal illness occurred among persons who had attended a museum fund-raiser in Wilmington, Delaware and an intercollegiate football game in Philadelphia. A survey of four groups attending these events showed that 31% (191/614) became ill. Altogether, those who consumed ice were 12 times more likely to experience either vomiting or diarrhea than those who did not (attack rate, 55% vs. 4%, P less than .001). Ice consumed at the events was traced to a manufacturer in southeastern Pennsylvania whose wells had been contaminated when flooded by a nearby creek after a torrential rainfall on 8 September. Of 19 affected persons tested within 1 week of exposure, 13 (68%) had at least a fourfold rise in antibody titer to the Norwalk virus. This report, the first to document an association of contaminated commercial ice with Norwalk gastroenteritis should prompt reassessment of government regulation of the production and distribution of ice.

Astrovirus and adenovirus associated with diarrhea in children in day care settings.

The relative importance of astrovirus and adenoviruses as etiologic agents of diarrhea among children in day care was examined. Stool specimens from this prospective study were screened for both astrovirus and adenovirus hexon with two new indirect double-antibody assays and for enteric adenoviruses with an EIA specific for serotypes 40 and 41. Astrovirus was detected in a significantly greater percentage of children with diarrhea (4%, 21/524) than of those without (less than 1%, 1/138) (P less than .05); however, no difference between such such children with adenovirus infections was found (8%, 43/565, and 8%, 10/129, respectively). Overall, 30% (13/43) of all adenovirus hexon-positive specimens were enteric serotypes, and by extrapolation, enteric adenoviruses were identified in an equal percentage of children (2%) with and without diarrhea. This study documents the presence of astrovirus and enteric adenoviruses among children in day care in the United States, associates astrovirus with diarrhea in this setting, and suggests that viral agents may be the most common enteric pathogens among children with diarrhea in day care.

Waterborne outbreak of Norwalk virus gastroenteritis at a southwest US resort: role of geological formations in contamination of well water.

From April 17 to May 1, 1989, gastroenteritis developed in about 900 people during a visit to a new resort in Arizona, USA. Of 240 guests surveyed, 110 had a gastrointestinal illness that was significantly associated with the drinking of tap water from the resort's well (relative risk = 16.1, 95% confidence interval 14.5 to 17.8) and this risk increased significantly with the number of glasses of water consumed (p less than 0.005). Three of seven paired sera tested for antibodies to the Norwalk agent had a four-fold or greater rise in titre. Water contaminated with faecal coliforms was traced back to the deep water well, which remained contaminated even after prolonged pumping. Effluent from the resort's sewage treatment facility seeped through fractures in the subsurface rock (with little filtration) directly into the resort's deep well. Although the latest technology was used to design the resort's water and sewage treatment plants, the region's unique geological conditions posed unexpected problems that may trouble developers faced with similar subsurface geological formations and arid climatic conditions in many parts of the world. In these areas, novel solutions are needed to provide adequate facilities for the treatment of sewage and supply of pure drinking water.

Outbreak of human calicivirus gastroenteritis in a day-care center in Sydney, Australia.

Between January and March 1988, an outbreak of gastroenteritis occurred among children and staff at a day-care center in Sydney, New South Wales, Australia. Over an 11-week period, 53 persons had 101 episodes of gastroenteritis; some patients had 5 separate episodes. The principal etiologic agent in the outbreak, human calicivirus (HCV), was detected by electron microscopy in 32% of fecal specimens from children and staff members with symptoms but in only 8% of asymptomatic individuals (P less than 0.01). HCV was confirmed by both an enzyme immunoassay and solid-phase immune electron microscopy. HCV infection was a particular problem in infants, who had the highest age-specific attack rates, had the greatest symptomatic/asymptomatic infection ratio, and were most likely to have a second symptomatic episode. The mode of transmission of this virus was not identified, and extensive efforts to control the 11-week outbreak had little effect. Prolonged excretion of HCV by some symptomatic patients and high rates of asymptomatic infection may have contributed to the extended duration of the outbreak. HCV may be a common cause of gastroenteritis in children that is under-recognized because of insensitive methods of detection.

Temporal synthesis of proteins and RNAs during human astrovirus infection of cultured cells.

Astroviruses are nonenveloped particles with a distinctive star-shaped surface structure that have been detected by electron microscopy in stool samples from humans and animals with gastroenteritis. We examined the patterns of macromolecular synthesis in astrovirus-infected cells with a goal of establishing a molecular basis for taxonomic classification. Trypsin is required for continuous replication of astrovirus in cultured cells; however, during a single cycle of infection, astrovirus antigen was synthesized earlier and at higher levels when serum, rather than trypsin, was included in the growth medium. This enhanced production of antigen, as measured by enzyme immunoassay, was accompanied by the appearance of aggregates of virus particles in the cytoplasm of infected cells. During astrovirus replication in cells cultured in the presence of serum, we detected a single infection-specific protein (90 kDa) beginning at 12 h postinfection. This protein was recognized by antiastrovirus rabbit serum and was sensitive to trypsin digestion in vitro, with the concomitant appearance of three smaller immunoreactive proteins (31, 29, and 20 kDa). We also detected two dactinomycin-resistant RNAs (7.2 and 2.8 kb), both of which were polyadenylated, in the cytoplasm of astrovirus-infected cells. The larger of these two RNAs is presumably the viral genome, whereas the smaller species may be a subgenomic messenger. Comparison of the proteins and RNAs synthesized in astrovirus-infected cells with those of the recognized families of nonenveloped single-stranded RNA animal viruses suggests that astroviruses should not be classified as members of either Caliciviridae or Picornaviridae.

Norwalk virus antigen and antibody response in an adult volunteer study.

To better define the optimum timing of specimen collection and identify alternate ways to diagnose Norwalk virus outbreaks, we looked at the timing of the antibody response and virus excretion in a human volunteer study. The Norwalk virus antibody titers and antigen in stool specimens were examined by biotin-avidin immunoassay. Our data suggest that in epidemic situations, convalescent-phase sera could be collected as soon as 13 days after the onset of illness and acute-phase sera could be collected as late as 5 days after onset. Our data also suggest that if sufficient serum samples are collected, convalescent-phase case and control serum samples can be used to identify Norwalk virus outbreaks. Antigen detection was much less sensitive than seroconversion for detecting infection.

Detection of Norwalk virus antibodies and antigen with a biotin-avidin immunoassay.

Biotin-avidin immunoassays (BAIs) were developed to detect Norwalk virus antigen and to measure Norwalk virus antibody. The BAI detected Norwalk virus infections by a fourfold titer rise in antibody in sera or by antigen in stool, with a sensitivity similar to or greater than that of the radioimmunoassay (RIA), and the BAI appeared to be more sensitive than the RIA for detecting antibody in single serum specimens. The BAI antigen test detected Norwalk antigen in all stools that were positive by the RIA, and in two stools negative by the RIA. Of 106 serum pairs tested by both the RIA and BAI, 94 demonstrated seroconversion, or lack of seroconversion, in both assays; 12 gave discordant results. Titers by the BAI generally exceeded those by the RIA by two- or fourfold. The BAI had advantages over the RIA in that it had added sensitivity in the detection of Norwalk antibody, was less time consuming, did not require frequent labeling of antibody, and did not have the problems associated with the use of isotopes.