Evaluation of a new enzyme-linked immunosorbent assay test for rotavirus antigen in faeces.

A new commercial test for the diagnosis of rotavirus gastroenteritis was assessed. With some modifications it compared favourably with electron microscopy and immunofluorescence.

Diagnostic electron microscopy of faeces. I. The viral flora of the faeces as seen by electron microscopy.

A method is described for examining viruses in faeces by direct electron microscopy using negative staining. The particles found in a group of patients with gastroenteritis and a group with other conditions are compared. Small particles in the range of sizes covering parvoviruses and enteroviruses were found about as frequently in each group. Many of these were probably bacteriophages. Many bacteriophages with tails of various sizes and lengths were found. Adenoviruses were found in five of eight patients from whom they were isolated. Reovirus- (or orbivirus-) like particles were found associated with some gastroenteritis patients.

Diagnostic electron microscopy of faeces. II. Acute gastroenteritis associated with reovirus-like particles.

Virus particles resembling reoviruses or orbiviruses were found in the faeces of 40 of 73 patients under 6 years of age with acute gastroenteritis and in faeces of only two babies among 31 patients under 6 years admitted to hospital with other diagnoses. In morphology the particles resemble orbiviruses more closely than reoviruses, but differ in appearance from the orbiviruses in having a smooth, circular outline with a well marked continuous rim as seen in negatively stained preparations. They appear not to be serologically related to reovirus types 1, 2, or 3 and may be members of a new group.

Diagnosis of rotavirus infection by cell culture.

Rotaviruses were detected by electronmicroscopy in 35 of 84 specimens of faeces from infants with diarrhoea, and in 31 by fluorescent staining of tissue cultures infected with help of centrifugation. LLC-MK2 cells were found to be the most sensitive, although primary and secondary human-embryo-kidney and primary calf-kidney cells could also be used. A micromodification of the tissue-culture method provides a relatively simple technique for the diagnosis of rotavirus infection, for the titration of virus infectivity and for estimating neutralising antibodies in serum.

Transmission of human rotaviruses to gnotobiotic piglets.

Faecal filtrates containing rotavirus particles, from children with acute infectious diarrhoea, were inoculated intranasally into gnotobiotic piglets. The piglets developed no symptoms, but birus was readily found by electron microscopy in their faeces during three serial passages. Among 11 piglets tested 3 weeks after inoculation of virus, all had developed fluorescent antibodies against tissue-culture-adapted calf rotavirus but only two had neutralising antibody. Growth of human rotavirus did not occur in either normal or "nude", thymus-deficient suckling mice.

Isolation of influenzavirus A and B in PLC/PRF/5 cells.

Virus isolation was attempted in PLC/PRF/5 cells, a continuous primary liver carcinoma line, and secondary rhesus monkey kidney (2 degrees RhMK) cells from 19 clinical samples from which influenza A (n = 4) and B (n = 15) had been isolated previously. Virus growth was determined by haemadsorption and immunofluorescence. Influenza A was recovered in both cell types from all four samples tested. Influenza B was re-isolated in both cell types from eight specimens, in PLC/PRF/5 only from two, and in 2 degrees RhMK only from one; no virus was recovered from the remaining four. Cultures of either virus were haemadsorption- and immunofluorescence-positive by six days post-inoculation. Cytopathic effect produced by the influenza A isolates in PLC/PRF/5 was minimal following initial inoculation of specimens, but increased on subsequent passage. Influenza B isolates produced a cytopathic effect in PLC/PRF/5 similar in extent and speed of development to that in 2 degrees RhMK. These results suggest that PLC/PRF/5 may be a suitable continuous cell line, and an alternative to monkey kidney cells, for the isolation and monitoring of antigenic variation of influenza viruses.

Diagnosis of adenovirus conjunctivitis by enzyme immunoassay.

The diagnosis of adenovirus conjunctivitis using a commercial enzyme immunoassay (EIA) was investigated and compared with virus isolation in cell culture. Of 53 eye swabs previously shown to be adenovirus-positive, EIA was positive with 33 and isolation in PLC/PRF/5 cells was successful with 49. Isolates were typed by serum neutralisation. Adenovirus types 3 and 4 were most common, with 14 and 12 isolates respectively. Nine isolates were found to be type 8 and eight were classified as untyped following unsuccessful neutralisation by antisera to types 1-8 and 14. The potential of EIA, and possible modifications to increase sensitivity, are discussed.

Adenovirus-associated gastro-enteritis in the north-west of England: 1991-1994.

Faeces, positive for adenoviruses by electron microscopy on initial examination at Manchester Public Health Laboratory (PHL) or Preston PHL during the period 1991-1994, were examined by an enzyme immunoassay which differentiated Ad40, Ad41 and non-AdF strains. Isolation and serotyping by neutralisation of non-AdF strains were attempted in PLC/PRF/5 cells. Of 452 specimens examined, 99 (22%) contained Ad40, 209 (46%) Ad41 and 144 (32%) non-AdF serotypes. From 1991 to 1993 the ratio of Ad40 to Ad41 was constant at approximately 1 to 1.6, but in 1994 it was almost 1 to 5. Non-AdF serotypes, of which Ad1 and Ad2 were the most common, accounted for 44% of specimens tested from 1991, but this dropped to 20% of those from 1994. An age/sex analysis showed that half the specimens examined were from infants under one year old, and that overall the male/female ratio was 3 to 2. The significance of these results is discussed, including the changing prevalence of Ad40 and Ad41, and the role of non-AdF serotypes in gastro-enteritis.

Isolation of enteroviruses and adenoviruses in continuous simian cell lines.

The ability of five simian cell lines--BGM, LLC-MK2, MA104, Vero and BSC-1--to isolate enteroviruses and adenoviruses from faeces was assessed and compared with that of primary or secondary rhesus monkey kidney (RhMK) and HEp2 cultures. For enteroviruses, LLC-MK2 and BGM were best of the five lines, detecting 77% and 73% respectively of 88 strains isolated, but neither was as good as RhMK, which detected 88%. Both Vero and BSC-1 detected less than 50%. All cell types were good at isolating poliovirus and coxsackie B strains, although LLC-MK2 and BGM were slightly better than MA104, Vero, BSC-1 and HEp2 for the latter. Greatest variation in sensitivity occurred with coxsackie A9 and echovirus isolates, with all lines being less sensitive than RhMK. However, the combined isolation rate for BGM, LLC-MK2 and HEp2 was the same as that for RhMK and HEp2, although the strains not detected by each cell combination were different. Both Vero and BSC-1 were reasonable for detecting adenoviruses, but neither was as good as HEp2. The reasons for variations in results, and the future use for such cell lines for isolating viruses from clinical and environmental samples, are discussed.

Epidemic viral enteritis in a long-stay children's ward.

Two outbreaks of acute gastroenteritis occurred in 1974 in a long-stay children's ward. Electron microscopy demonstrated rotaviruses in faeces from the affected children in the first outbreak, and adenoviruses in faeces from affected children and a nurse in the second outbreak. The illness in both outbreaks was very mild; but the diarrhoea associated with rotavirus infection usually lasted 5-8 days (in one patient it lasted for 28 days) and sometimes started with vomiting; whereas the adenovirus-associated diarrhoea lasted only 2-4 days and was not associated with vomiting. Neither the rotaviruses nor the adenoviruses could be established in tissue-culture.

Rotavirus enteritis in the West Midlands during 1974.

During 1974 in the West Midlands of England, 38% of children less than 6 years old with enteritis were excreting rotaviruses. Children aged from 6 months to 3 years were those most commonly infected. Rotavirus infections were most common during winter with only a few sporadic cases during summer. A possible pathogen was detected in 59% of patients examined.

Pathology of rotavirus infection in suckling mice: A study by conventional histology, immunofluorescence, ultrathin sections, and scanning electron microscopy.

Pathologic changes induced in the small intestine of suckling mice by rotavirus infection were studied by conventional histology, immunofluorescence, scanning electron microscopy, and electron microscopy of ultrathin sections. Infection could be detected within 24 hours in a few mice, but after 2 days it was well established. Swollen, often vacuolated infected cells were found on the sides and tips of villi from which they rapidly became detached; microvilli showed variable irregularity. Immature enterocytes from crypts replaced lost infected cells. By the tenth day very few infected cells could still be found. Both tubular structures and spherical particles occurred in the infected cells. Only tubular structures were found in nuclei.

Intestinal damage in rotavirus and adenovirus gastroenteritis assessed by d-xylose malabsorption.

The absorption of D-xylose infused into the duodenum was assessed in infants with acute gastroenteritis. 1-hour blood-xylose levels were low in 6 patients found to harbour rotavirus in the small intestinal aspirate. Normal levels (greater than 1-26 mmol/l) were obtained in the absence of virus particles in the small intestine in a further 6 patients: in 3 of these adenovirus or rotavirus was recovered from the stools. Three patients with adenovirus in the small intestinal juice and ill with acute gastroenteritis also had low xylose levels. This finding supports earlier epidemiological studies that adenovirus may be a causative agent of acute infantile gastroenteritis. The association between virus in the small intestine and xylose malabsorption may indicate mucosal damage. Formal proff of this is awaited.