Failure of cooking to prevent shellfish-associated viral gastroenteritis.

BACKGROUND: In January 1995, Florida experienced the largest outbreak of oyster-associated gastroenteritis ever reported. METHODS: We interviewed both the cohort of persons from 38 gatherings where illness was reported and a sample of harvesters and harvest-area residents. Oysters were traced by means of tags and dealer records, and water quality measures in harvest areas were reviewed. We examined stool specimens for small round structured viruses by means of electron microscopy and amplification of RNA by reverse-transcriptase polymerase chain reaction. We also tested serum specimens for antibodies to Norwalk virus. RESULTS: Of 223 oyster eaters, 58% (129/223) became ill, compared with 3% (2/76) of non-oyster eaters (relative risk, 22; 95% confidence interval, 5.6-87.0). Most oyster eaters (67% [149/223]) ate only cooked (grilled, stewed, or fried) oysters. Oyster eaters who reported eating only thoroughly cooked oysters were as likely to become ill as those who ate raw oysters (relative risk, 0.68; 95% confidence interval, 0.45-1.0; P = .1). In 29 clusters, implicated oysters were from Apalachicola Bay, Florida. A community outbreak occurred in 2 bayside communities before the oyster harvest, leading to an increase in the reportedly common practice of overboard dumping of feces. Small round structured viruses were identified in the stool specimens of 2 harvest-area residents and 9 persons from 8 clusters. Results of water quality tests for fecal coliforms were within acceptable limits. CONCLUSIONS: This large outbreak of gastroenteritis associated with oysters may have resulted from overboard dumping of feces during a community outbreak of diarrheal illness. Our findings of acceptable water quality measures for fecal contamination and the lack of appreciable protective effect from cooking leave the consumer with no assurance of safety.

Use of stool collection kits delivered to patients can improve confirmation of etiology in foodborne disease outbreaks.

BACKGROUND: In 68% of foodborne disease outbreaks, no etiologic pathogen is identified. In two-thirds of outbreaks with no identified etiology, no stool specimens are submitted for testing. METHODS: From April 2001 to March 2003, we pilot-tested use of prepackaged, self-contained stool specimen collection kits in 3 states, delivered to and from patients by courier or mail, to improve rates of specimen collection in the outbreak setting. Specimens were tested for bacterial and viral pathogens at health department laboratories, and results were correlated with epidemiological investigation data. RESULTS: Specimens were returned by > or =1 person in 52 (96%) of 54 outbreaks in which kits were deployed; in total, 263 (76%) of 347 persons who received kits returned specimens. Resolution of symptoms was the most commonly cited reason for nonsubmission of kits. An etiology was confirmed in 37 (71%) of 52 outbreaks with specimens returned; 28 (76%) were attributable to norovirus, and 9 (24%) were attributed to bacterial pathogens. Stool kits were well received and cost an average of approximately 43 dollars per specimen returned. CONCLUSIONS: In two-thirds of foodborne disease outbreaks in which delivered stool collection kits were successfully deployed, an etiologic organism was identified. Delivery of kits to and from patients to improve rates of stool collection in outbreaks in which specimens might otherwise not be submitted could substantially reduce the number of outbreaks with an unknown etiology.

Capsid protein composition of reference strains and wild isolates of human astroviruses.

Astroviruses are small RNA viruses that are frequently associated with gastroenteritis in humans and animals. Despite much work on the genetic analysis of astrovirus strains, little progress has been made in the characterization of the proteins composing mature virions. We have analyzed the capsid protein composition of the reference strains and several wild isolates of human astroviruses using high-resolution polyacrylamide gradient gels. For reference strains of the seven serotypes analyzed, a consistent pattern of three infection-specific proteins--designated P1, P2, and P3 -was generally observed. The strains could be divided into two groups, based upon the reactivity of these proteins in immune precipitation assays that used homologous rabbit serum. One group included reference types 1 4 for which all three proteins were precipitated by homologous rabbit sera; for the other group, types 5 7, only proteins P2 and P3 were precipitated. When wild isolates from around the world were compared to the reference strains, a correlation between genetic type and the pattern of protein sizes and immune reactivity was observed for strains of the common types (1-4). Strains of types 2 and 4 consistently exhibited P3 proteins larger than those of types 1 and 3. Unusual patterns of proteins or immune reactivity were detected in strains of types 5-7, indicating that there may be incomplete processing of the capsid precursor during growth in cell culture.

Molecular epidemiology of human astroviruses.

Human astroviruses (HAstVs) are associated with 5-9 percent of cases of gastroenteritis in young children. Seven serotypes (HAstV-1 to -7), which correlate with genotypes, have been defined by using immune typing methods. We have used partial nucleotide sequence information from the capsid protein gene for molecular typing of 29 unique human astrovirus strains obtained from prospective studies of children with gastroenteritis in Egypt and Malawi. HAstV-1 was the most commonly detected strain, consistent with previous studies, but a surprising variety of strains were identified in both collections. An eighth astrovirus type, HAstV-8, has been defined on the basis of the complete capsid protein gene sequence and was detected in both collections analysed in this study. Although HAstV-8 and HAstV-4 strains segregate into well resolved clades by analysis of sequences from the region encoding protein P2 (VP32), the pair-wise distances between these types are less than those between strains of the other serotypes. In contrast, analysis of sequences from the region encoding protein P3 unambiguously resolve HAstV-4 and HAstV-8 strains, consistent with their classification as distinct serotypes. Overall, strains representing six of the eight serotypes were detected in two collections of samples from prospective studies of gastroenteritis in young children indicating that multiple astrovirus types are frequently co-circulating within communities.

Gastroenteritis viruses: an overview.

Acute gastroenteritis is among the most common illnesses of humankind, and its associated morbidity and mortality are greatest among those at the extremes of age, children and the elderly. In developing countries, gastroenteritis is a common cause of death in children < 5 years that can be linked to a wide variety of pathogens. In developed countries, while deaths from diarrhoea are less common, much illness leads to hospitalization or doctor visits. Much of the gastroenteritis in children is caused by viruses belonging to four distinct families--rotaviruses, caliciviruses, astroviruses and adenoviruses. Other viruses, such as the toroviruses, picobirnaviruses, picornavirus (the Aichi virus), and enterovirus 22, may play a role as well. Viral gastroenteritis occurs with two epidemiologic patterns, diarrhoea that is endemic in children and outbreaks that affect people of all ages. Viral diarrhoea in children is caused by group A rotaviruses, enteric adenoviruses, astroviruses and the caliciviruses; the illness affects all children worldwide in the first few years of life regardless of their level of hygiene, quality of water, food or sanitation, or type of behaviour. For all but perhaps the caliciviruses, these infections provide immunity from severe disease upon reinfection. Epidemic viral diarrhoea is caused primarily by the Norwalk-like virus genus of the caliciviruses. These viruses affect people of all ages, are often transmitted by faecally contaminated food or water, and are therefore subject to control by public health measures. The tremendous antigenic diversity of caliciviruses and short-lived immunity to infection permit repeated episodes throughout life. In the past decade, the molecular characterization of many of these gastroenteritis viruses has led to advances both in our understanding of the pathogens themselves and in development of a new generation of diagnostics. Application of these more sensitive methods to detect and characterize individual agents is just beginning, but has already opened up new avenues to reassess their disease burden, examine their molecular epidemiology, and consider new directions for their prevention and control through vaccination, improvements in food and water quality and sanitary practices.

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 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.

RT-PCR identification and typing of astroviruses and Norwalk-like viruses in hospitalized patients with gastroenteritis: evidence of nosocomial infections.

BACKGROUND: Astroviruses (HAstVs) and 'Norwalk-like viruses' (NLV) are frequent causes of gastroenteritis worldwide, though no data on the strains in circulation or their prevalence is available for France. OBJECTIVES: We applied molecular methods to detect HAstVs and NLVs by reverse transcription-polymerase chain reaction (RT-PCR) in fecal samples collected during a 2-year period from children and adults hospitalized with gastroenteritis. STUDY DESIGN: All samples negative for rotavirus and adenovirus by latex agglutination which contained small (25-40 nm) viral particles observed by electron microscopy (EM) were examined by RT-PCR. RT-PCR products were sequenced to characterize the HAstV and NLV strains present. RESULTS: A total of 75 samples were analyzed by RT-PCR, of which 15 were positive for HAstV and 24 for NLV. Several distinct strains of serotype 1 HAstV, the predominant serotype, circulated during the period. Nineteen of the 24 NLVs were of the G2 genogroup including Mexico-like (n=10), Bristol-like (n=8), and Hawaii-like viruses (n=1); two were genogroup 1. Overall, seven (47%) of the 15 HAstV infections and nine (37.5%) of the 24 NLV infections appeared to be nosocomially acquired based on the date of admission in hospital and the date of illness. CONCLUSION: This study provides additional evidence of the importance of nosocomial infections caused by NLV and HAstV.

An outbreak of viral gastroenteritis in a nursing home: importance of excluding ill employees.

BACKGROUND: In May 1994, 43 persons in a nursing home were reported with gastroenteritis. An outbreak investigation was conducted to determine risk factors for gastroenteritis among residents and staff. METHODS: Data were analyzed using contingency tables; relative risks (RR) and statistical significance were determined with Fisher's Exact Test. The chi-squared statistic to perform a goodness of fit test for the binomial distribution was used to determine whether cases occurred randomly and independently of each other. Stools were tested for bacterial enteric pathogens, ova, and parasites and were examined by electron microscopy, Southern hybridization, and reverse transcription-polymerase chain reaction. Paired sera were collected to detect fourfold rises in antibody titer by enzyme immunoassay against Norwalk viruses. RESULTS: Of 121 residents, 62 (51%) had gastroenteritis, as did 64 (47%) of the 136 staff. The index case was a nurse who became ill at work and continued to work, while symptomatic, for another 2 days. Only residents who had received medications from this nurse between May 17 and May 20 became ill on the first day of the outbreak (13 of 35 versus 0 of 5). Nurses and nurse aides were more likely than employees without direct resident contact to be cases (46 of 68 versus 18 of 58; RR, 2.18; P < .001). Bacterial stool cultures and parasite examinations were negative. Results of electron microscopy, polymerase chain reaction with Southern hybridization, and enzyme immunoassay indicated the causative agent was a small, round, structured virus similar to the Snow Mountain Agent. CONCLUSION: To minimize outbreaks in nursing homes, we recommend that ill staff be excluded from work until symptoms resolve.

An outbreak of acute gastroenteritis caused by a small round structured virus in a geriatric convalescent facility.

An outbreak of acute gastroenteritis (AGE) occurred in a 201-bed geriatric convalescent facility in Los Angeles County during December 1988 through January 1989. The attack rate was 55% among residents and 25% among employees. Illnesses were characterized by vomiting and diarrhea to a lesser extent, and the absence of fever. Bacterial and parasitic tests in a sample of patients were negative. A 27 nm small round structured virus (SRSV) was identified in one of 30 stools studied by immune electron microscopy (IEM). While rotavirus and influenza A and B were found in three, one and three cases, respectively, no alternative etiologic agent could be demonstrated for most cases. The outbreak met Centers for Disease Control (CDC) clinical and epidemiologic criteria for Norwalk-like gastroenteritis. The death rate of residents was not elevated beyond baseline during the outbreak; however, one healthy employee had diarrhea and dehydration and died after developing an arrhythmia. An autopsy showed moderate, diffuse lymphocytic and neutrophilic myocarditis, and viral studies found influenza A in left ventricular tissue. Fourteen (25%) of 57 employee cases worked in occupations without routine stool or patient contact. At least nine of these employees lacked evidence of direct fecal contact, and transmission of infection in these cases may have been airborne.

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.

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.

The changing epidemiology of astrovirus-associated gastroenteritis: a review.

Our understanding of the epidemiology of astrovirus-associated gastroenteritis has changed markedly with each improvement in detection method. In early surveys based on electronmicroscopy (EM), astroviruses appeared to be a rare cause of gastroenteritis, being found in fewer than 1% of children with diarrhea, usually in small outbreaks of disease and primarily during the winter season. The development and use of monoclonal antibodies and enzyme immunoassays (EIA) to detect astroviruses led to reports of a higher prevalence (2.5%-9%) of astrovirus infection among patients hospitalized with diarrhea. Astroviruses appeared second only to rotaviruses as a cause of hospitalization for childhood viral gastroenteritis. Studies based on EIA detection of astroviruses indicate that astroviruses are common causes of diarrhea in children worldwide, and that most children are infected during their first two years of life. The elderly and the immunocompromised represent high-risk groups as well. The observations that newborns monitored prospectively rarely have repeat disease and that the rate of detection decreases with increasing age suggest that immunity to astroviruses, as immunity to rotaviruses, may develop early in life. The cloning and sequencing of astroviruses have led to more sensitive assays to detect the viruses by reverse transcription, polymerase chain reaction (RT-PCR). Application of RT-PCR for detection of astroviruses in children in day-care centers showed a marked increase in the detected prevalence of astrovirus-associated diarrhea, the rate of asymptomatic infection, and the duration of shedding of virus among those infected, when compared with studies that used other methods. As with rotaviruses, neither the mode of transmission nor the reservoir of astrovirus infection has been identified. Both immune and molecular-based assays to detect astrovirus serotypes indicate that serotype 1 is most common worldwide, although the predominant serotypes may vary by region and time. In the absence of obvious strategies to prevent astrovirus-associated diarrhea, vaccines might be considered if further studies establish that the disease burden would render such a vaccine cost-effective.

Optimization of extraction and PCR amplification of RNA extracts from paraffin-embedded tissue in different fixatives.

A method was developed for fast and efficient isolation of RNA from paraffin-embedded tissue sections for subsequent PCR analysis. This method is based on the binding of RNA to acid-treated glass beads in the presence of a high molarity of guanidinium salt. It can be completed within an hour, and obviates the need for dewaxing and phenol/chloroform extractions. The effect of various fixatives and fixation times was tested and the amplification of actin mRNA fragments ranging in length from 82 to 507 bp was used to demonstrate the presence of RNA in the extracts. The method was compared to existing extraction techniques by studying the quality of the templates for reverse-transcriptase polymerase chain reaction amplification (RT-PCR), using virus-infected and mock-infected paraffin-embedded cell pellets as a model. PCR amplification of cellular and viral RNA was successful for RNA isolated by use of all extraction techniques, although the glass bead method was preferred for its simplicity and rapidity. Specimens fixed with formalin were found to be suitable for PCR, but the best results were obtained with acetone-fixed paraffin-embedded material. Dewaxing of tissue sections had no effect on the yield and quality of RNA extractions, and further purification of the extracts using gel filtration did not improve the results. After the protocols were optimized, rotavirus-infected cell pellets were used to demonstrate that extraction and amplification of dsRNA was possible. The information obtained from the studies with the model system was used for extraction of toroviral and rotaviral RNA from archival intestinal material. These data indicate that paraffin-embedded archival tissue can be used for RT-PCR analysis, adding an important technique to diagnostic pathology and retrospective studies.

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.

Outbreak of norovirus illness associated with a swimming pool.

On 3 February 2004, the Vermont Department of Health received reports of acute gastroenteritis in persons who had recently visited a swimming facility. A retrospective cohort study was conducted among persons attending the facility between 30 January and 2 February. Fifty-three of 189 (28%) persons interviewed developed vomiting or diarrhoea within 72 h after visiting the facility. Five specimens tested positive for norovirus and three specimen sequences were identical. Entering the smaller of the two pools at the facility was significantly associated with illness (RR 5.67, 95% CI 1.5-22.0, P=0.012). The investigation identified several maintenance system failures: chlorine equipment failure, poorly trained operators, inadequate maintenance checks, failure to alert management, and insufficient record keeping. This study demonstrates the vulnerability of recreational water to norovirus contamination, even in the absence of any obvious vomiting or faecal accident. Our findings also suggest that norovirus is not as resistant to chlorine as previously reported in experimental studies. Appropriate regulations and enforcement, with adequate staff training, are necessary to ensure recreational water safety.

Molecular analysis of noroviruses involved in acute gastroenteritis outbreaks in military units in Israel, 1999-2004.

The study presented here was conducted to determine the genetic properties of noroviruses (NoVs) identified between 1999 and 2004 in army recruits with acute gastroenteritis. Partial sequence analysis of the RNA-dependent RNA polymerase gene revealed the presence of two major sub-genogroups, all of which were related to genogroup II of NoV. Serological analysis using recombinant antigens confirmed this observation. Local strains associated with a 1999 outbreak were closely related to GII-6 strains, while those identified later were very closely related to GII-4 strains. GII-4 strains were also associated with an outbreak in civilian nursing homes in Israel in 2002 and samples from this outbreak were included in this study for comparison. This is the first report describing the molecular properties of NoV strains associated with diarrhea-related morbidity in Israel.

Common and distinct regions of defective-interfering RNAs of Sindbis virus.

Defective-interfering (DI) particles are helper-dependent deletion mutants which interfere specifically with the replication of the homologous standard virus. Serial passaging of alphaviruses in cultured cells leads to the accumulation of DI particles whose genomic RNAs are heterogeneous in size and sequence composition. In an effort to examine the sequence organization of an individual DI RNA species generated from Sindbis virus, we isolated and sequenced a representative cDNA clone derived from a Sindbis DI RNA population. Our data showed that: (i) the 3' end of the DI RNA template was identical to the 50 nucleotides at the 3' end of the standard RNA; (ii) the majority (75%) of the DI RNA template was derived from the 1,200 5'-terminal nucleotides of the standard RNA and included repeats of these sequences; and (iii) the 5' end of the DI RNA template was not derived from the standard RNA, but is nearly identical to a cellular tRNAAsp (S. S. Monroe and S. Schlesinger, Proc. Natl. Acad. Sci. U.S.A. 80:3279-3283, 1983). We have also utilized restriction fragments from cloned DNAs to probe by blot hybridization for the presence of conserved sequences in several independently derived DI RNA populations. These studies indicated that: (i) a 51-nucleotide conserved sequence located close to the 5' end of several alphavirus RNAs was most likely retained in the DI RNAs; (ii) the junction region containing the 5' end of the subgenomic 26S mRNA was deleted from the DI RNAs; and (iii) the presence of tRNAAsp sequences was a common occurrence in Sindbis virus DI RNAs derived by passaging in chicken embryo fibroblasts.

RNAs from two independently isolated defective interfering particles of Sindbis virus contain a cellular tRNA sequence at their 5' ends.

Defective interfering (DI) particles are deletion mutants that interfere specifically with the replication of homologous standard virus. We have determined the 5'-terminal nucleotide sequences of two DI RNA populations by the following methods: (i) cloning of the cDNA from one of the DI RNA populations and sequencing a representative clone, and (ii) using both DI RNA populations as templates for preparing primer-directed cDNA transcripts and sequencing these transcripts. The 5' terminal sequences of the two DI RNA populations were not derived from standard Sindbis viral RNA but were almost identical to those of a cellular tRNAAsp.