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

"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.

A prospective case-control study of the role of astrovirus in acute diarrhea among hospitalized young children.

This study examines the importance of astroviruses as a cause of acute diarrhea in hospitalized children <10 years old during a 5-year period. Stools were screened by electron microscopy and were tested for astrovirus, rotavirus, and enteric adenovirus by EIA. During the study, 14.6% of hospitalized children had diarrhea. Astroviruses were second only to rotaviruses as etiologic agents of both community-acquired and nosocomial diarrhea. Community-acquired astrovirus infection occurred in 6.8% of patients, and nosocomial disease occurred in 16.2%. Most cases occurred from March through June, and astrovirus type 1 was the most common. The symptoms of astrovirus-infected children were similar to those of children with rotavirus infection. However, astrovirus-infected children had a lower median age, less dehydration, and lower symptom severity scores and were less likely to have been admitted for gastroenteritis than were children with rotavirus. Astrovirus, for which only rehydration therapy is required, should be considered as another common diarrheal pathogen in children <2 years old.

"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.

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.

An outbreak of acute gastroenteritis in a geriatric long-term-care facility: combined application of epidemiological and molecular diagnostic methods.

OBJECTIVE: To assess possible transmission modes of, and risk factors for, gastroenteritis associated with Norwalk-like viruses (NLVs) in a geriatric long-term-care facility. METHODS: During a prolonged outbreak of acute gastroenteritis, epidemiological data on illness among residents and employees were collected in conjunction with stool, vomitus, and environmental specimens for viral testing. NLVs were identified by electron microscopy in stool and vomitus specimens, and further characterized by reverse-transcriptase polymerase chain reaction and nucleotide sequencing. Potential risk factors were examined through medical-record review, personal interview, and a self-administered questionnaire sent to all employees. RESULTS: During the outbreak period, 52 (57%) of 91 residents and 34 (35%) of 90 employees developed acute gastroenteritis. Four case-residents were hospitalized; three residents died at the facility shortly after onset of illness. A point source was not identified; no association between food or water consumption and gastroenteritis was identified. A single NLV strain genetically related to Toronto virus was the only pathogen identified. Residents were at significantly higher risk of gastroenteritis if they were physically debilitated (relative risk [RR], 3.5; 95% confidence interval [CI95], 1.0-12.9), as were employees exposed to residents with acute gastroenteritis (RR, 2.6; CI95, 1.1-6.5) or ill household members (RR, 2.3; CI95, 1.4-3.6). Adherence to infection control measures among the nursing staff may have reduced the risk of gastroenteritis, but the reduction did not reach statistical significance. CONCLUSIONS: In the absence of evidence for food-borne or waterborne transmission, NLVs likely spread among residents and employees of a long-term-care facility through person-to-person or airborne droplet transmission. Rapid notification of local health officials, collection of clinical specimens, and institution of infection control measures are necessary if viral gastroenteritis transmission is to be limited in institutional settings.

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.

Prevalence of astrovirus infection among Chilean children with acute gastroenteritis.

The frequency of astrovirus infection in 456 Chilean children with diarrhea was determined by enzyme-linked immunosorbent assay, reverse transcriptase PCR, and cell culture. Astrovirus was detected in 16.5% of rotavirus-negative and 7% of rotavirus-positive samples obtained from emergency rooms or hospitals and in 11% of samples from day care centers. HAst-1 was the predominant serotype identified.

A prospective study of astrovirus diarrhea of infancy in Mexico City.

AIM: To describe the epidemiologic and clinical characteristics of astrovirus-associated diarrhea in a cohort of young children from a periurban community in Mexico City. METHODS: From November, 1988, through December, 1991, a total of 214 children were enrolled in a longitudinal study of diarrhea and monitored from birth to 18 months of age. A stool specimen was collected during each episode of diarrhea. Specimens from a total of 510 diarrhea episodes were tested for astrovirus by enzyme immunoassay and examined for other enteric pathogens. The antigenic types of astrovirus were determined by a typing enzyme immunoassay. RESULTS: Astrovirus was detected in 26 (5%) of 510 diarrhea episodes, with an incidence rate of 0.1 episode/child year; the highest rate was in children 13 to 18 months of age. Astrovirus-associated diarrhea was characterized by a median of 4 stools (range, 2 to 10) during the first 24 h, a median duration of 3 days (range, 1 to 21), vomiting (20%), and fever (7%). No cases of dehydration or repeat symptomatic infections were observed. Coinfection with another pathogen was detected in 11 of the 26 episodes (42%). Serotype 2 (35%) was most common, followed by serotypes 4 (15%), 3 (11%), and 1 and 5 (4% each); 31% were nontypable. Astrovirus-associated diarrhea was less severe, as measured by the number of stools (4.3 +/- 1.9), than diarrhea caused by rotavirus (7.1 +/- 2.8) or when coinfections occurred (5.5 +/- 1.6; P = 0.008). CONCLUSIONS: Astrovirus was associated with 5% of the episodes of diarrhea in this cohort of young Mexican children and presented as a mild secretory diarrhea. Five predominant antigenic types were detected with type 2 being the most common.

Astrovirus infection in association with acute, persistent and nosocomial diarrhea in Bangladesh.

BACKGROUND: Diarrhea is an important public health concern in developing countries such as Bangladesh. Diarrhea in children that persists for 14 days or more occurs in 7% of patients in Bangladesh and frequently results in death. Astrovirus has been demonstrated as a cause of acute and nosocomial diarrhea and can be excreted for prolonged periods, yet its importance as a cause of diarrhea among children in a developing country like Bangladesh has not been investigated. METHODS: We tested 629 stool specimens from patients with acute diarrhea, 153 from patients with persistent diarrhea, 175 specimens from 76 patients hospitalized for diarrhea who were sampled repeatedly to detect nosocomial infection and 428 from nonhospitalized healthy children (controls). All children enrolled in the study were <5 years of age. Astrovirus was detected by enzyme immunoassay and other enteropathogens were detected by standard techniques. RESULTS: The detection of astrovirus increased significantly with the duration of diarrhea. Astrovirus was found in 23 (15%) specimens from patients with persistent diarrhea, 26 (4%) patients with acute diarrhea, but only 8 (2%) healthy controls. This trend remained when we limited our analysis to infants <12 months of age and to episodes in which astrovirus was the sole pathogen. Among patients with nosocomial diarrhea, 16% of postadmission specimens were positive for astrovirus when the admission specimen was negative. CONCLUSION: The observation that astrovirus is detected more frequently with diarrhea of increasing duration suggests the need for further studies to determine whether astrovirus plays a causative role in persistent diarrhea or is a secondary agent.

A viral gastroenteritis outbreak associated with person-to-person spread among hospital staff.

OBJECTIVE: To identify the etiologic agent and risk factors associated with a hospital ward outbreak of gastroenteritis. SETTING: A regional referral hospital in upstate South Carolina. METHODS: We reviewed patient charts, surveyed staff, and tested stool from acutely ill persons. A case was defined as diarrhea and vomiting in a staff member or patient from January 5 to 13, 1996. RESULTS: The initial case occurred on January 5 in a staff nurse who subsequently was hospitalized on the ward and visited by many staff colleagues. The staff were at a significantly greater risk for gastroenteritis than were patients (28/89 [31%] vs 10/91 [11%]; relative risk [RR], 2.9; 95% confidence interval [CI95], 1.5-5.5). All 10 case-patients had been exposed to case-nurses (assigned nurses who were primary caretakers), and eight had documented exposure to case-nurses 1 to 2 days before their illness. Patients exposed to case-nurses had a significantly increased risk of illness (8/57 [14%] vs 0/32; RR, >4.5; CI95, undefined). Neither staff nor patients had significantly increased risk from food, water, ice, or exposure to case-patients. Electron microscopy identified small round-structured viruses (SRSVs) in nine of nine stool samples. CONCLUSION: This nosocomial outbreak of gastroenteritis was likely caused by SRSVs introduced by a staff member and spread via person-to-person transmission from and among staff. The potential for spread of SRSV-associated gastroenteritis from and among staff should be considered in developing strategies to prevent similar outbreaks in hospital settings.

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.

Parkville virus: a novel genetic variant of human calicivirus in the Sapporo virus clade, associated with an outbreak of gastroenteritis in adults.

This report describes the characterization of Parkville virus, the etiologic agent of an outbreak of foodborne gastroenteritis, that has the morphology of a calicivirus and genetic properties that distinguish it from previously identified strains in the Sapporo/Manchester virus clade. Sequence analysis of the Parkville virus genome showed it contained the RNA-dependent RNA polymerase motifs GLPSG and YGDD characteristic of members of the family Caliciviridae with an organization identical to that reported for the Manchester virus where the capsid region of the polyprotein is fused to the RNA polymerase. Parkville virus however, demonstrates considerable sequence divergence from both the Manchester and Sapporo caliciviruses, providing the first indications that genetic diversity exists within caliciviruses of this previously homogeneous clade. On the basis of recent advances in the genetic characterization of members of the family Caliciviridae, we propose a new interim phylogenetic classification system in which Parkville virus would be included with Manchester and Sapporo virus as a separate group distinct from the small round-structured viruses (Norwalk-like viruses) that also cause diarrhea in humans.

A one-tube method of reverse transcription-PCR to efficiently amplify a 3-kilobase region from the RNA polymerase gene to the poly(A) tail of small round-structured viruses (Norwalk-like viruses).

Amplification of a 3-kb genome region from the RNA polymerase gene to the 3' poly(A) tail of small round-structured virus (SRSV) by reverse transcription-PCR (RT-PCR) has been difficult to achieve because of a stable secondary structure in a region between the RNA polymerase gene and the 5' end of the second open reading frame. We have developed a one-tube RT-PCR method to efficiently amplify this region. The method comprises three procedures: purification of poly(A)+ RNA from a starting RNA solution by oligo(dT)30 covalently linked to latex particles, buffer exchange, and continuous RT and PCR in a single tube containing all reaction components. The key elements of this method are (i) first-strand cDNA synthesis with the Superscript II version of RNase H- Moloney murine leukemia virus reverse transcriptase at 50 degrees C for 10 min by using the RNA-oligo(dT)30 hybrid on the latex particles as the template and primer, and (ii) PCR by Taq and Pwo DNA polymerases mixed together with a mixture of 12 phased oligo(dT)25 antisense primers. The detection threshold of the one-tube RT-PCR method was as little as 0.2 ng of the crude RNA used as the source of the template. Using this method, we obtained 3-kb products from 24 SRSV strains previously characterized into four genetic groups. These included 5 P1-A, 4 P1-B, 5 P2-A, and 10 P2-B strains. Because SRSVs have not yet been cultivated in vitro, this novel method should facilitate molecular characterization of SRSVs to provide a firm scientific foundation for improvements and refinements of SRSV diagnostics.

Correlation of patient immune responses with genetically characterized small round-structured viruses involved in outbreaks of nonbacterial acute gastroenteritis in the United States, 1990 to 1995.

Small round-structured viruses (SRSVs) are a genetically and antigenically diverse group of caliciviruses that are the most common cause of outbreaks of acute nonbacterial gastroenteritis. We have applied both molecular techniques to characterize SRSVs in fecal specimens and serologic assays using four different expressed SRSV antigens to examine the distribution of outbreak strains in the United States and determine if the immune responses of patients were strain specific. Strains from 23 outbreaks of SRSV gastroenteritis were characterized by reverse transcription-PCR and nucleotide sequencing of a 277-base region of the capsid gene. These strains segregated into two distinct genogroups, I and II, comprising four and six clusters of strains respectively, each representing a distinct phylogenetic lineage. Serum IgG responses in patients were measured by enzyme immunoassay using expressed capsid antigens of Norwalk virus (NV), Toronto virus (TV), Hawaii virus (HV), and Lordsdale virus (LV), representing four of the 10 clusters. While strains in genogroups I and II were antigenically distinct, within genogroups, the specificity of the immune response varied greatly. Patients infected with genogroup I strains which had as much as 38.5% aa divergence from NV demonstrated relatively homologous seroresponses to the single NV antigen. In contrast, in genogroup II, homologous seroresponses to TV and HV were only present when the infecting strains showed less than 6.5% aa divergence from these antigens. These results suggest that TV and HV represent not only separate genetic clusters in genogroup II but also separate antigenic groups, each of which is related but distinguishable. In addition, two genetically distinct SRSV strains were identified for which we have no homologous antigen. This study suggests that while current molecular diagnostics are capable of detecting the full range of SRSVs, additional expressed antigens will be required to detect an immune response to SRSV infection caused by all the antigenically diverse strains.

A university outbreak of gastroenteritis due to a small round-structured virus. Application of molecular diagnostics to identify the etiologic agent and patterns of transmission.

An epidemiologic investigation of a gastroenteritis outbreak in December 1994 indicated that salad consumption during lunch was linked with illness on 2 days (5 December: odds ratio [OR]=3.1, 95% confidence interval [CI]=2.0-5.0; 6 December: OR=3.1, 95% CI=1.9-4.9). Single stool or vomitus specimens from ill students and staff (case-patients) were examined for bacterial and viral pathogens. Small round-structured viruses (SRSVs) were detected by electron microscopy in stool specimens from 9 of 19 case-patients and in vomitus specimens from 3 of 5 case-patients. By reverse transcription-polymerase chain reaction (RT-PCR), the SRSVs were shown to be G-2/P2-B type strain. The nucleotide sequences of RT-PCR products from vomitus and stool specimens of ill students were identical to stool specimens from the ill salad chef. These findings suggest that a single SRSV strain was the etiologic agent in the outbreak that was possibly transmitted to students through consumption of contaminated salad. Epidemiologic investigation in conjunction with molecular diagnostics may enable early identification of sources of infection and improve outbreak control.

Characterization of Toronto virus capsid protein expressed in baculovirus.

Toronto virus (TV), previously called "minireovirus", a human calicivirus classified as genogroup 2 and phylogenetic type P2-A, was originally described in association with diarrhea in children. The second open reading frame, encoding the capsid protein of TV24, was expressed in a baculovirus recombinant. The recombinant baculovirus produced a protein (rTV) with an apparent molecular mass of 58 kDa that self-assembled into virus-like particles approximately 30 nm in diameter with a density of 1.29 g/ml. Antigenic and immunogenic characteristics of these particles were determined by protein immunoblot, immunoprecipitation, and enzyme immunoassay. Seroconversion to the rTV protein was detected in 6 of 8 (75%) patients from a recent outbreak of gastroenteritis associated with a virus of similar phylogenetic type. These results confirm and extend the previous reports of the expression of the Norwalk and Mexico virus capsid proteins.

Epidemiologic applications of novel molecular methods to detect and differentiate small round structured viruses (Norwalk-like viruses).

The molecular epidemiology of a large, multistate outbreak of oyster-associated gastroenteritis [Kohn et al. (1995): Journal of the American Medical Association 273:466-471. Dowell et al. (1995): Journal of Infectious Diseases 171:1497-1503.] was examined using new methods to detect small round structured viruses (SRSVs) by reverse transcription-polymerase chain reaction (RT-PCR) and to characterize strains by Southern hybridization and nucleotide sequencing of 81-bp of a PCR product amplified from the RNA polymerase gene. Of 37 stool specimens examined from patients in eight clusters of the multistate outbreak, 32 (86%) gave RT-PCR products specific for SRSVs of P1-A phylogenetic group. Nineteen PCR products from the eight clusters were confirmed to have the identical sequence, indicating that this large outbreak was attributed to a single strain of SRSV. In one of the eight clusters, five (63%) of eight patients had a mixed infection with a second SRSV strain that belonged to P2-B phylogenetic group. Of 12 specimens from patients in five other outbreaks and one sporadic case which occurred at the same time as the multistate outbreak, 10 (83%) gave products specific for SRSVs representing four phylogenetic groups (P1-A, P1-B, P2-A, and P2-B). The sequences of the P1-A products from two outbreaks and that of the P2-B product from another outbreak were identical to the P1-A sequence from the eight clusters and the P2-B sequence from the one cluster of the multistate outbreak, respectively. These results demonstrate the first application of these methods to enhance our understanding of the molecular epidemiology of SRSVs and provide answers of public health interest that could not have been obtained using classical epidemiologic methods alone.

Typing of human astroviruses from clinical isolates by enzyme immunoassay and nucleotide sequencing.

A typing enzyme immunoassay (TYPE-EIA) was used to determine the antigenic types of 64 astrovirus-positive specimens from nine collections from seven countries. Six of the seven known astrovirus types were detected in the collections, with HAstV-1 predominating in all collections for one from the United Kingdom. Selected specimens were analyzed further by reverse transcriptase PCR and nucleotide sequencing of 348 bp within the capsid protein precursor region of the genome. The phylogenetic groupings (genotypes) determined from the sequences were entirely consistent with the antigenic groupings (serotypes) of isolates obtained by using the TYPE-EIA. The genetic variation within genotypes was small compared with the variation between genotypes, allowing unambiguous categorization of all specimens. Although some strains from widely separated geographic areas had identical sequences, in general, within a region most strains of the same type were identical. The TYPE-EIA may help further our understanding of the epidemiology of astrovirus and the possible role of serotype-specific immunity, while further knowledge of sequences could facilitate the development of simpler molecular methods of typing astrovirus strains.

Impact of rotavirus infection on a paediatric hospital in the east end of London.

AIMS: To study the impact of confirmed rotavirus infection at a paediatric hospital; to use the data to obtain a minimum estimate of the cost of treating reported cases of rotavirus in England and Wales. METHODS: Data were obtained on all patients with rotavirus over a two year period. Information was collected on 386 patients with rotavirus infection who were treated at the 120 bed Queen Elizabeth Hospital for Children in East London. This included the virus serotype, the patient's age, whether they required intravenous infusion, duration of hospital stay, numbers of patients treated in the casualty department, and numbers who had to be admitted. Treatment costs were obtained from the Finance Department of the Hospitals for Sick Children. RESULTS: The minimum cost of treating patients, excluding the cost of medical staff at the hospital, was estimated to be 95,400 pounds a year. One hundred and forty eight (38%) patients were admitted to the wards and a further 49 patients developed symptoms while in hospital. Intravenous infusion was required by 18 patients. The mean duration of hospital stay was 5.5 days. One hundred and eighty nine (49%) patients were treated with oral rehydration solution in casualty, given advice, and sent home. Ninety four per cent of the patients were aged under 2 years. The findings were comparable with those obtained in a study at Texas Children's Hospital, USA. The G serotype (VP7) of rotavirus did not influence the severity of infection. CONCLUSION: Rotavirus infections accounted for a significant number of patients treated in casualty, admissions to hospital, and bed occupancy in a paediatric hospital. The estimated cost of treating reported cases of rotavirus in England and Wales is in excess of 6.3 pounds million a year.