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.

Efficacy of two doses of RIT 4237 bovine rotavirus vaccine for prevention of rotavirus diarrhoea.

Candidate oral bovine rotavirus vaccine RIT 4237 or placebo was given to 252 Finnish infants at birth and at 7 months of age. No vaccine-associated reactions were observed. Primary rotavirus ELISA IgM responses were detected in 36% of the infants after the first vaccination; after the second dose 68% of the vaccinees were seropositive for rotavirus ELISA IgG antibody. The infants remained in clinical follow-up over two rotavirus epidemic seasons (total 28 months). Counted from child years in follow-up the overall vaccine protection rate was 43%. The clinical severity of rotavirus episodes was assessed using a numerical score 0-20. Vaccine protection rate for cases with a score greater than or equal to 7 was 57% and for cases with a score greater than or equal to 11 it was 89%. It is concluded that vaccination with a bovine rotavirus vaccine at birth and at 7 months of age, with the second dose given shortly before rotavirus epidemic season, protects infants against moderately severe and severe rotavirus diarrhoea in the first 2 years of life.

Evaluation of RIT 4237 bovine rotavirus vaccine in newborn infants: correlation of vaccine efficacy to season of birth in relation to rotavirus epidemic period.

A single oral dose of bovine rotavirus vaccine RIT 4237 or placebo was given to 2 groups of 5-day-old infants, born in October 1984 (n = 244) and June 1985 (n = 245), who remained in follow-up for 2.8 and 2.0 years, respectively. The vaccine had no effect on the total number of detectable episodes of rotavirus diarrhoea: there were 22 cases in the vaccinees and 24 in the placebo recipients in the October group and 18 and 16 respectively in the June group. However, vaccination decreased significantly the clinical severity of rotavirus diarrhoea, as assessed by a numerical score 0-20; this vaccine effect was much greater in the infants born in October. The mean severity scores for vaccine and placebo recipients were 4.55 and 10.75 respectively in the October group (p less than 0.0001, t-test) and 8.2 and 11.6 respectively in the June group (p = 0.010, t-test). Vaccine-induced clinical protection against rotavirus diarrhoea did not correlate well with serological response after vaccination, but showed good correlation to the presence of rotavirus antibodies before the rotavirus epidemic season. It is concluded that bovine rotavirus vaccine is more efficacious when given immediately before the rotavirus epidemic season: the vaccine effect may be amplified by exposure to wild rotaviruses during the season.

Temporal and geographical distributions of human rotavirus serotypes, 1983 to 1988.

Between 1983 and 1988, subgroups and serotypes were determined for 907 of 1,084 clinical specimens of rotaviruses collected in various countries of Europe, North and South America, Africa, and Asia. Enhanced enzyme immunoassays based on monoclonal antibodies specific for rotavirus proteins VP6 and VP7 were used. Significant differences in the prevalent serotypes were detected from year to year in the United Kingdom and Brazil and also in different countries during the same year. Throughout the study, rotavirus serotype 1 was detected most often (53.8%), followed in frequency by serotype 2 (17.8%), serotype 3 (12.1%), serotype 4 (11.1%), and serotypes other than 1 to 4 (5.1%). No individual serotype was found to predominate consistently in any one location. In the United Kingdom, rotavirus serotypes varied in prevalence in a regular but not predictable way. We suggest that a similar epidemiology might be found in other settings. Seventeen unusual strains were detected. Of these, five strains did not react with reference monoclonal antibodies specific for subgroup I and subgroup II, but they reacted with rotavirus group A-specific polyclonal and monoclonal antibodies; four strains were of subgroup II, serotype 2, and at least one had a "long" electropherotype; two strains were of subgroup I, serotype 2 with a long electropherotype; and one strain was of subgroup I, serotype 3. Five group C rotaviruses were detected.

Comparative evaluation of the WHO and DAKOPATTS enzyme-linked immunoassay kits for rotavirus detection.

Faeces obtained from 1,163 children (including 66 newborn babies) were analysed in parallel for the presence of rotavirus particles using two enzyme-linked immunosorbent assay kits. The kits had been formulated by the WHO Collaborating Centre for Reference and Research on Rotavirus (WHO-ELISA kit) and by DAKOPATTS (DAKO-ELISA kit) to be suitable for use in laboratories in developing countries. The kits were evaluated in laboratories in Burma, Chile, India, Mexico, Pakistan, Sri Lanka and the United Kingdom. Comparison of the results obtained with the two kits indicated that the DAKO-ELISA had an overall sensitivity of 97% and a specificity of 97% relative to the WHO-ELISA. In individual laboratories the DAKO-ELISA (K349) kit had a sensitivity in the range 90-100%, and a specificity of 85-100%. The kit showed a sensitivity of 100% and a specificity of 98% in assays on faeces obtained from newborn babies. We conclude that the DAKO-ELISA is as sensitive and specific as the WHO-ELISA for the detection of rotavirus antigen in faeces.

Recent advances in the study of viral gastroenteritis.

Acute infectious gastroenteritis is the commonest cause of death in children under 5 years old who live in developing countries. In developed countries, whilst deaths occur rarely, gastroenteritis remains an important public health problem. Before the early 1970s the cause of the majority of diarrhoeal disease episodes was a mystery. The recognized causes of infectious diarrhoea at the time were bacteria and parasites. During the 1970s a number of previously unknown viruses were discovered and subsequently shown to cause infectious gastroenteritis. Numerous studies which were conducted during the late 1970s and early 1980s confirmed the world-wide significance of these viruses as important pathogens, especially the rotaviruses, which can now be cultivated. Emphasizing recent progress, an overview is given of the virus, its pathogenesis and vaccine strategies for preventing disease. Other viruses, including adenoviruses, caliciviruses and astroviruses, are also described and their significance assessed.

Comparative evaluation of the WHO and DAKOPATTS enzyme-linked immunoassay kits for rotavirus detection

Faeces obtained from 1163 children (including 66 newborn babies) were analysed in parallel for the presence of rotavirus particles using two enzyme-linked immunosorbent assay kits. The kits had been formulated by the WHO Collaborating Centre for Reference and Research on Rotavirus (WHO-ELISA kit) and by DAKIPATIS (DAKO-ELISA kit) to be suitable for use in laboratories in developing countries. The kits were evaluated in laboratories in Burma, Chile, India, Mexico, Pakistan, Sri Lanka and the United Kingdom. Comparison of the results obtained with the two kits indicated that the DAKO-ELISA has an overall sensitivity of 97 per cent and a specificity of 97 per cent relative to the WHO-ELISA. In individual laboratories the DAKO-ELISA (K349) kit had a sensitivity in the range 90-100 per cent in assays on faeces obtained from newborn babies. We conclude that the DAKO-ELISA is as sensitive and specific as the WHO-ELISA for the detection of rotavirus antigen in faeces(AU)

A virus with a bisegmented double-stranded RNA genome in rat (Oryzomys nigripes) intestines.

Examination of the intestinal contents of free-living Oryzomys nigripes rats by PAGE revealed two sharply defined bands that could be stained by ethidium bromide or by silver nitrate with comparable intensities. The molecules forming these bands were susceptible to digestion by pancreatic RNase A but not by RNase T1 or by DNase I. Their lengths were estimated to be about 2.6 and 1.5 kbp, respectively, by comparison with rotavirus SA11 genome segments. They cosedimented in CsCl gradients at a density of 1.39 to 1.40 g/ml, together with uniform particles approximately 35 nm in diameter with indistinct surface structure. It is suggested that these particles represent an as yet undescribed virus with a bisegmented double-stranded RNA genome, for which the name 'picobirnavirus' is proposed.

Subgroups, serotypes, and electrophoretypes of rotavirus isolated from children in Bangui, Central African Republic.

The subgroups and serotypes of 178 strains of rotavirus isolated from diarrheic and healthy children in Bangui, Central African Republic, during a 27-month period were determined by enzyme-linked immunosorbent assay. The subgroup was determined for 152 of the viral strains, 18.4% being subgroup I and 81.6% being subgroup II. Of the 143 strains which could be serotyped, 71.3% were serotype 1, 15.4% were serotype 2, and 13.3% were serotype 3. Serotypes 1 and 3 were detected throughout the study, while serotype 2 was detected only during 8 months. No serotype exhibited any special epidemiological properties. The serotypes were found to consist of three different electrophoretypes, two long ones (A and B) and a short one (C). All subgroup I, serotype 2 strains presented short electrophoretypes. Strains with identical long electrophoretypes A were either serotype 1 or serotype 3.

Epidemiology of rotavirus subgroups and serotypes in Belem, Brazil: a three-year study.

Rotavirus subgroups and serotypes were determined in 61 rotavirus-positive faecal samples obtained from children living in Belém, Brazil, followed up from birth to 3 years of age. Fifty-five (90%) of the specimens were subgrouped and the serotypes of 30 (49%) of them were determined. Subgroup II was detected in 49 (89%) of the 55 subgrouped strains. Serotype 1 was present in 15 (50%) of the 30 serotyped samples; serotypes 2, 3 and 4 were found in 30%, 3.3% and 16.7% respectively, of these specimens. Absence of Vp7, the major outer capsid glycoprotein, did not allow serotyping in 21 (34.4%) of the 61 rotavirus-positive specimens, and an unidentifiable new serotype was found in faeces of one child. In addition, 4 samples were classified as subgroup II serotype 2 (which is very unusual). Twelve (80%) of the 15 serotype 1 (subgroup II) specimens were collected from children (5 of them asymptomatic) during their first year of life. All 9 serotype 2 (subgroups I, II, or not determined) samples were detected during the second and third years of life, 7 (77.8%) of them were related to apparent infections. The 5 serotype 4 (subgroup II) samples were obtained throughout the study period, and were associated with both symptomatic (3 cases) and asymptomatic infections. Thirteen children had more than 1 rotavirus infection. Three had 3 successive infections. In 3 cases, the initial infection (either symptomatic or asymptomatic) caused by serotype 1, was followed by a subsequent diarrhoeic episode associated with serotype 2.

Detection of Breda virus antigen and antibody in humans and animals by enzyme immunoassay.

Enzyme immunoassays were developed for the detection of Breda virus antibody and antigen. Cattle sera collected in the United Kingdom were found to have a high prevalence of antibody (55%) to Breda virus when examined in a competitive enzyme-linked immunosorbent assay. A low prevalence of antibody was found in pigs (2.2%), and no antibody was found in sheep or goat sera. No antibody to either Breda virus or Berne virus was detected in human sera collected from veterinarians and farm workers. Only 1 of 430 human fecal specimens (0.2%) contained Breda virus antigen detectable by enzyme-linked immunosorbent assay.

Prevalence of antibody to group B (atypical) rotavirus in humans and animals.

Enzyme-linked immunosorbent assays were developed for the detection of group B rotavirus antigen and antibody. The specificities of both assays were evaluated for antigens and serum specific for rotavirus groups A to D. Serum collected in the United Kingdom from different animal species exhibited the following high prevalence of group B rotavirus-specific antibody: pigs, 97%; cattle, 71%; sheep, 91%; and goats, 91%. In human serum, a lower prevalence of group B-specific antibody was detected; serum from blood donors showed 10% prevalence, and serum from veterinarians showed 4% prevalence.

A new family of vertebrate viruses: Toroviridae.

The proposed family Toroviridae is characterized by eveloped, peplomer-bearing particles containing an elongated tubular nucleocapsid with helical symmetry. The capsid may bend into an open torus, conferring a biconcave disk or kidney-shaped morphology to the virion (largest diameter 120-140 nm) or the capsid may be straight, resulting in a rod-shaped particle (35 X 170 nm). Morphogenesis is by budding of preformed nucleocapsids through membranes mainly of the Golgi system and of the rough endoplasmic reticulum. Berne virus, which is proposed as the family prototype, contains a single strand of infectious positive-sense RNA, Mr about 6.5 X 10(6), which is polyadenylated. The RNA is surrounded by the major nucleocapsid phosphoprotein (Mr about 20,000) which, in turn, is enveloped by a membrane containing one major protein (Mr 22,000) and a phosphoprotein (Mr 37,000). The viral peplomers, about 20 nm long, carry determinants for neutralization and hemagglutination; they are formed by a polydisperse N-glycosylated protein (Mr 75,000-100,000). Four major subgenomic polyadenylated RNAs have been identified in infected cells, with Mrs of 3.0, 0.71, 0.46 and 0.26 X 10(6). Torovirus replication is inhibited by actinomycin D, alpha-amanitin and pre-irradiation of the host cell with UV light. All toroviruses identified so far cause enteric infections and are probably transmitted by the fecal-oral route. Serologic relationships between equine, bovine and human toroviruses have been demonstrated.

The diagnostic gap in diarrhoeal aetiology.

It is well established that rotaviruses of group A are the most important cause of severe diarrhoea in children. The causes of most cases of infectious diarrhoea still remain unidentified, however, and there must be other viruses to be found. 'Novel' rotaviruses have recently been discovered, mainly in animals (serogroups B and C in pigs and humans, D in birds, and one or more further groups in sheep and other mammals). Except for the group B virus which has caused widespread outbreaks of quite severe diarrhoea in adults in China (still not reported from outside China) these novel rotaviruses are rarities in the human and probably represent uncommon zoonotic infections. We speculate that the Chinese virus might have arisen by reassortment of genetic segments of animal group B viruses or perhaps by mutation, and so became infectious for man. The problem of identifying and determining the importance of small round viruses is reviewed. It seems likely that the group of small, round structured viruses, including Norwalk and the viruses of plainly calicivirus morphology, are all representatives of a whole group of enteric caliciviruses. Until they can be more easily cultivated it will be difficult to make diagnostic reagents available to all. Astroviruses and genuine parvoviruses have been found by many people in many countries. Astroviruses are probably more important as pathogens in lambs than in children or calves; parvoviruses can only be established as significant in epidemics. The coronavirus-like particles, first found in Vellore and Bristol, are still enigmatic and their role in diarrhoea is uncertain. Toroviridae, recently discovered as causes of diarrhoea in ungulates, do not seem to be at all important as causes of diarrhoea in humans. Possibly fruitful approaches to future searches are outlined: firstly to make more extensive use of immuno-electron microscopy; and secondly to try to improve existing tissue culture systems to make them more sensitive to enteric viruses.

Effects of two negative staining methods on the Chinese atypical rotavirus.

The Chinese atypical (Group B) rotavirus, previously called the adult diarrhoea rotavirus (ADRV), was examined by transmission electron microscopy using either uranyl acetate or potassium phosphotungstate (PTA) as negative stains. Complete rotavirus particles were seen using uranyl acetate which were indistinguishable morphologically from typical rotaviruses. In the same preparations virus particles with differing degrees of degradation were seen after staining with PTA. This effect was not related to pH of the PTA and could be prevented by fixation of the specimen by 0.1 per cent glutaraldehyde. It is concluded that the use of PTA can give rise to falsely negative results for specimens containing this virus.

Preliminary characterisation of torovirus-like particles of humans: comparison with Berne virus of horses and Breda virus of calves.

Pleomorphic virus-like particles have been observed by electron microscopy in the faeces of children and adults with diarrhoea. Some of these particles were approximately 100 nm in diameter and had a "fringe" of closely applied peplomers approximately 10 nm long; they closely resembled Berne virus of horses and Breda virus of calves, the two representatives of a newly proposed family called the Toroviridae. In one sample a toroidal nucleoprotein-like structure was observed within the particles. For two samples a buoyant density of 1.14 g/ml was determined by centrifugation through a sucrose density gradient. One sample possessed a haemagglutinin for rat erythrocytes. The serological relationship between these different viruses was observed by immune electron microscopy, haemagglutination inhibition, and serum neutralisation. The role of these virus-like particles as candidate pathogens of humans is discussed.