Biomedical subjects

S S Monroe

Publications and source records attributed to S S Monroe.

At least 73 records · Page 4Linked to original sources

PubMed1994

Norwalk virus-associated gastroenteritis traced to ice consumption aboard a cruise ship in Hawaii: comparison and application of molecular method-based assays.

Investigation of an outbreak of acute nonbacterial gastroenteritis on a cruise ship provided an opportunity to assess new molecular method-based diagnostic methods for Norwalk virus (NV) and the antibody response to NV infection. The outbreak began within 36 h of embarkation and affected 30% of 672 passengers and crew. No single meal, seating, or food item was implicated in the transmission of NV, but a passenger's risk of illness was associated with the amount of ice (but not water) consumed (chi-square for trend, P = 0.009). Of 19 fecal specimens examined, 7 were found to contain 27-nm NV-like particles by electron microscopy and 16 were positive by PCR with very sensitive NV-specific primers, but only 5 were positive by a new highly specific antigen enzyme immunoassay for NV. Ten of 12 serum specimen pairs demonstrated a fourfold or greater rise in antibody titer to recombinant baculovirus-expressed NV antigen. The amplified PCR band shared only 81% nucleotide sequence homology with the reference NV strain, which may explain the lack of utility of the fecal specimen enzyme immunoassay. This report, the first to document the use of these molecular method-based assays for investigation of an outbreak, demonstrates the importance of highly sensitive viral diagnostics such as PCR and serodiagnosis for the epidemiologic investigation of NV gastroenteritis.

Antibodies, Viral↗

PubMed1994

Application of PCR to detect Norwalk virus in fecal specimens from outbreaks of gastroenteritis.

Norwalk virus (NV) and other small round-structured viruses (SRSVs) are frequent causes of gastroenteritis outbreaks. The recent cloning and sequencing of the NV genome has made it possible to detect NV and Norwalk-related viruses from fecal specimens by reverse transcription (RT)-PCR. We applied this technique to the examination of a total of 139 fecal specimens from 19 outbreaks characterized by NV serology, including 56 samples from 7 NV outbreaks, 36 from 6 Norwalk-related virus outbreaks, and 47 from 6 outbreaks with SRSVs visualized by electron microscopy that were serologically unrelated to NV. Three primer pairs were evaluated: two pairs in the polymerase region of NV and one pair near the 3' end of the genome. When one set of primers (primer pair 51-3) from the polymerase region was used, 40% of all samples were positive by RT-PCR and specimens from the NV outbreaks were more likely to be positive (64%) than those from outbreaks associated with Norwalk-related viruses (44%) or SRSVs (8%). To determine the relationship of the outbreak strains to NV, we compared the sequences of a 145-base portion of the polymerase gene from 10 specimens obtained from five different outbreaks characterized as NV by serology. No two outbreak strains had the same sequence in this 145-base portion of the polymerase gene, and the identities of the nucleotide and amino acid sequences of these products compared with the sequences of the corresponding region of NV ranged from 62 to 79% and 69 to 90%, respectively. Because of sequence diversity in the polymerase region, the successful application of RT-PCR to investigations of outbreaks of suspected NV-associated gastroenteritis will depend on the use of either multiple primer pairs or primers made against regions of the genome that are more conserved.

Amino Acid Sequence↗

PubMed1994

Characterization of a variant strain of Norwalk virus from a food-borne outbreak of gastroenteritis on a cruise ship in Hawaii.

A gastroenteritis outbreak affecting at least 217 (41%) of 527 passengers on a cruise ship was caused by a variant strain of Norwalk virus (NV) that is related to but distinct from the prototype NV strain. Consumption of fresh-cut fruit served at two buffets was significantly associated with illness (P < or = 0.01), and a significant dose-response relationship was evident between illness and the number of various fresh-cut fruit items eaten. Seven (58%) of 12 paired serum specimens from ill persons demonstrated at least fourfold rises in antibody response to recombinant NV capsid antigen. A 32-nm small round-structured virus was visualized by electron microscopy in 4 (29%) of 14 fecal specimens, but none of the 8 specimens that were examined by an enzyme immunoassay for NV antigen demonstrated antigen. Four (40%) of 10 fecal specimens were positive by reverse transcriptase-PCR by using primer pairs selected from the polymerase region of NV. In a 145-bp region, the PCR product shared only 72% nucleotide sequence identity with the reference NV strain and 77% nucleotide sequence identity with Southampton virus but shared 95% nucleotide sequence identity with UK2 virus, a United Kingdom reference virus strain. In addition, the outbreak virus was serotyped as UK2 virus by solid-phase immune electron microscopy. The genetic and antigenic divergence of the outbreak strain from the reference NV strain highlights the need for more broadly reactive diagnostic assays and for improved understanding of the relatedness of the NV group of agents.

Antigenic Variation↗

PubMed1993

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.

Base Sequence↗

PubMed1993

Enteric viruses and diarrhea in HIV-infected patients. Enteric Opportunistic Infections Working Group.

BACKGROUND: Diarrhea occurs frequently among persons with the acquired immunodeficiency syndrome, but the cause often remains unknown. We used a group of diagnostic assays to determine which viruses were etiologic agents of diarrhea in a group of persons infected with the human immunodeficiency virus (HIV). METHODS: Stool and serum specimens were obtained from HIV-infected patients enrolled in a longitudinal study in Atlanta. Fecal specimens from patients with diarrhea and from control patients without diarrhea were screened by electron microscopy, polyacrylamide-gel electrophoresis, and enzyme immunoassays for rotaviruses, enteric adenoviruses, caliciviruses, picobirnaviruses, and astroviruses. Paired serum samples were tested for antibody responses to Norwalk virus and picobirnavirus. RESULTS: Viruses were detected in 35 percent of 109 fecal specimens from patients with diarrhea but in only 12 percent of 113 specimens from those without diarrhea (P < 0.001). Specimens from patients with diarrhea were more likely than those from patients without diarrhea to have astrovirus (12 percent vs. 2 percent, P = 0.003); picobirnavirus (9 percent vs. 2 percent, P = 0.017); caliciviruses, including small round structured viruses (6 percent vs. 1 percent, P = 0.062); and adenoviruses (9 percent vs. 3 percent, P = 0.047). They were also more likely to have a mixed viral infection (6 percent vs. 0 percent, P = 0.006). With the use of polyacrylamide-gel electrophoresis to analyze concentrated RNA extracts from stool, picobirnavirus was detected in fecal specimens from 6 of the 65 patients with diarrhea and was associated with prolonged viral shedding and chronic diarrhea. No rotaviruses, enteric adenoviruses, or instances of seroconversion to positivity for Norwalk virus were observed. CONCLUSIONS: Novel enteric viruses such as astrovirus and picobirnavirus may be more important etiologic agents of diarrhea in HIV-infected patients than previously recognized and may be more common than either bacterial or parasitic enteropathogens.

Adenoviruses, Human↗

PubMed1993

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.

Animals↗

PubMed1993

Outbreaks of astrovirus gastroenteritis in day care centers.

OBJECTIVE: This study evaluated astrovirus as a cause of diarrhea outbreaks among infants and toddlers in day care centers. DESIGN: Stool specimens were collected weekly during four periods (from January 1986 through December 1991) from children 6 to 30 months of age who were enrolled in prospective studies of diarrhea in day care centers. All diarrheal stool specimens were tested for bacterial enteropathogens, rotavirus, enteric adenovirus, and Giardia lamblia. A total of 1365 stool specimens from 70 outbreaks in which no etiologic agent was identified and from another 11 outbreaks with a known cause were tested for astrovirus, by means of a monoclonal antibody-based enzyme immunoassay. Confirmatory testing was performed by reverse transcriptase-polymerase chain reaction with primers designed to produce an 89 base-pair product. RESULTS: Astrovirus was detected in 6 (7%) of the 81 outbreaks. Of 217 children tested, 73 (34%) were infected with astrovirus; infections in 35 (48%) were symptomatic and in 38 (52%) asymptomatic. The six outbreaks lasted 11 to 44 days (median 22 days). Astrovirus excretion was detected for a duration of 2 to 30 days, with excretion occurring from 1 to 8 days (median 2 days) before diarrhea began to 1 to 20 days (median 2 days) after diarrhea ceased. Younger children (< or = 12 months) were at greater risk than older children (p = 0.011) of becoming infected with astrovirus during an outbreak and were more likely (p = 0.015) to have symptoms when infected. Of 24 specimens with astrovirus by enzyme immunoassay, 20 (83%) were confirmed to have the virus by reverse transcriptase-polymerase chain reaction. CONCLUSION: Astrovirus was an important cause of outbreaks of diarrhea among children attending day care centers, more frequently infected younger children, and often produced asymptomatic infections.

Age Factors↗

PubMed1993

Enzyme-linked immunosorbent assay reactivity of torovirus-like particles in fecal specimens from humans with diarrhea.

Toroviruses are recognized enteric pathogens of cattle and horses; in humans, similar pleomorphic particles have been described, but doubt has been raised concerning their identity as viruses. We screened fecal samples from humans with diarrhea for the presence of torovirus-like particles (TVLPs) by electron microscopy and subsequently used an enzyme-linked immunosorbent assay (ELISA) with bovine torovirus reference reagents to test for the presence of torovirus antigens. To add another selection criterion to this heterologous ELISA, we enriched the TVLPs from the stool specimens by using sucrose density gradients before testing. The results of ELISA and EM correlated significantly, the ELISA having a sensitivity of 68% and a specificity of 86% (chi-square, P < 0.0001). In the gradient, peaks of ELISA reactivity were found at a buoyant density of 1.16 g/ml and were parallel to those found when using bovine torovirus. Furthermore, in 50% of the ELISA-positive gradients, a hemagglutinin for human group O erythrocytes comigrated with the peaks of ELISA reactivity. We were unable to isolate human TVLPs in human colonic tumor or rectal tumor cells. We cloned and sequenced amplification products obtained by low-stringency polymerase chain reaction amplification using consensus primers mapping to the 3' end of the genome of animal toroviruses, but found no significant homologies with animals torovirus sequences. Rabbits were inoculated with material from the gradient peak fractions of human stool specimens, and their sera were assayed for immunologic comparison with bovine torovirus as a reference. A two-way antigenic cross-reactivity was seen between human TVLP and bovine torovirus reagents when tested by ELISA. The rabbit antisera to human TVLP detected a higher number of electron microscopy-positive stool specimens than did the rabbit antisera to bovine torovirus. The application of these assays and reagents should help to elucidate the roles of TVLPs and toroviruses in diarrheal disease in humans.

Animals↗

PubMed1993

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.

Antibodies, Viral↗

PubMed1993

Role of maturation cleavage in infectivity of picornaviruses: activation of an infectosome.

Maturation of picornaviruses involves assembly of a "provirion," which undergoes an autocatalytic cleavage of VP0 to VP2 plus VP4. RNA transcripts from a cDNA clone of human rhinovirus 14 mutated at asparagine 68, one of the residues in the maturation cleavage site, generated normal yields of 150S particles which were noninfectious in the plaque assay because they were unable to initiate a second cycle of infection. These cleavage-defective provirions were otherwise indistinguishable from mature virions in sedimentation coefficient, binding affinity to monoclonal antibodies against neutralization sites IA, II, and III, attachment to HeLa cell receptors, and rate of cell-mediated conformational changes to form 125S A-particles and 80S empty capsids. These results suggest that maturation cleavage is required for the function of a previously undescribed intermediate which transfers packaged RNA across the membrane and into the cytosol. For this hypothetical intermediate, we propose the name infectosome. Since the native virus has a particle/PFU ratio of about 800, such an intermediate will be difficult to find. Mutations at serine 10 in VP2 reduced maturation cleavage to a rate sufficiently slow to show that the infectivity of virus particles increased with the degree of cleavage of VP0 to VP4 and VP2. This article describes the first characterization of a pure form of a picornaviral provirion, and hence the first direct evidence that provirions of picornaviruses lack infectivity.

Amino Acid Sequence↗

PubMed1993

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.

Amino Acid Sequence↗

PubMed1992

Coding assignments of the genome of adult diarrhea rotavirus.

Adult diarrhea rotavirus (ADRV) has caused epidemics of diarrhea in China since 1982 and remains the only group B rotavirus associated with widespread disease in humans. We recently characterized the proteins of ADRV and have now proceeded to identify the gene segments encoding each protein. Viral RNA transcripts were synthesized in vitro with the endogenous viral RNA polymerase and separated by electrophoresis in agarose. The individual transcripts were translated in a cell-free system using nuclease-treated rabbit reticulocyte lysates. The translation products were compared with polypeptides found in purified virus and were characterized by SDS-PAGE, immunoprecipitation, and Western blot analysis using antisera to double- and single-shelled virions, virus cores, and monoclonal antibodies. Furthermore, individual RNA transcripts were hybridized to total dsRNA to determine their genomic origin. Based on this analysis, the core polypeptides VP1, VP2 and VP3 are encoded by segments 1, 2, and 3, respectively. The main polypeptides in the inner capsid, VP6, and the outer capsid, VP4 and VP7, are encoded by segments 6, 4, and 8 respectively. Segments 5, 7, and 9 code for 60, 45, and 30 kDa nonstructural polypeptides. Two other nonstructural polypeptides (24 and 25 kDa) are derived from gene segment 11. Gene segment 10 codes for a 26 kDa polypeptide that is precipitated with serum to ADRV and may be a structural protein VP9. With this exception, gene coding assignments of ADRV are comparable to those of the group A rotaviruses. Our results have clear implications for further work in cloning, sequencing, and expression genes of ADRV and can provide direction towards understanding the origin and the evolution of this virus.

Animals↗

PubMed1991

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.

Cohort Studies↗

PubMed1991

Electron microscopic reporting of gastrointestinal viruses in the United Kingdom, 1985-1987.

We examined some epidemiological features of the viruses associated with gastrointestinal illness, using national data reported by electron microscopists in the United Kingdom. During the 3 years analyzed (1985-1987), a total of 1,993 positive detections of astroviruses, caliciviruses, coronaviruses, and small round structured viruses (SRSVs) were reported. In 1 year of this period, 8,210 rotaviruses were reported. More than 90% of the astroviruses and caliciviruses were detected in children under 5 years of age, while coronaviruses and SRSVs were detected in adults as well as children. Detections of astroviruses increased in the winter and were infrequent during the summer, a seasonal pattern similar to that observed for rotaviruses. There was some variability between reporting regions in rates of detection of fecal viruses. We have attempted to identify the reasons for this. We make suggestions for improving the detection of human fecal viruses, and we recognize the need for continued surveillance of these agents.

Adolescent↗

PubMed1991

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.

Adenovirus Infections, Human↗

PubMed1991

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.

Antibodies, Viral↗

PubMed1991

Detection of astrovirus in pediatric stool samples by immunoassay and RNA probe.

Two new astrovirus assays, a rapid biotin-avidin enzyme immunoassay (EIA) and RNA probe hybridization, were developed and compared with an established astrovirus assay, an indirect EIA, and immune electron microscopy. Sensitivity and specificity were evaluated by using a screening panel of 22 astrovirus-positive and 305 astrovirus-negative fecal specimens. The biotin-avidin assay was equivalent in performance to the reference indirect assay, and both could detect about 10 ng of viral protein. Although the probe was more sensitive than either EIA and could detect higher dilutions of virus in tissue culture and stool specimens, it did not detect more astrovirus-positive fecal specimens. Of the 22 astrovirus-positive specimens detected by the EIAs, 20 were confirmed by immune electron microscopy with hyperimmune rabbit antiserum. To determine the usefulness of EIAs for large epidemiologic studies, EIAs were used to screen 1,289 stool specimens from three studies of children with and without diarrhea. Astrovirus was detected in 3.5% of specimens from children with diarrhea and 1.9% of specimens from those without diarrhea. Our results indicate that the biotin-avidin EIA is an efficient, sensitive, and specific method for routinely screening large numbers of fecal samples and that its application in epidemiologic studies may yield higher rates of astrovirus infection than have been found previously by other methods.

Avidin↗

PubMed1991

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.

Australia↗