Rapid detection of rotavirus in stool by latex agglutination: comparison with radioimmunoassay and electron microscopy and clinical evaluation of the test

A latex agglutination test (LX) using antisera prepared against Nebraska calf diarrhea virus (NCDV) is described for the detection of rotavirus in stool of children with acute gastroenteritis. The test was compared with electron microscopy (EM) and radioimmunoassay (RIA) with 100 stools positive or negative for rotavirus. Out of 53 stools positive in RIA or EM, 49 were positive in LX and 4 were negative. Two specimens negative in EM and RIA were falsely positive in LX. The method was also tested in two clinical series with 115 stools from 101 children. Altogether 67/115 stools were positive in RIA, and 62/115 in LX. Out of 7 stools with contradictory results, 6 were negative in LX but positive in RIA, and 1 was positive in LX but negative in RIA. The results indicate that the LX is suitable for rapid screening of rotavirus gastroenteritis in clinical practice.     

Solid-phase radioimmunoassay of IgA, IgG, and IgM antibodies to human rotavirus.

A solid-phase radioimmunoassay (RIA) has been developed for the detection of human rotavirus-specific IgA, IgG, and IgM antibodies. Nebraska calf diarrhea virus grown in LLC-MK2 cell cultures in the presence of trypsin was directly adsorbed onto polystyrene balls, and antibodies that attached to the virus-coated balls were detected by subsequent binding of 125I-labeled antibodies specific to human alpha, gamma or mu chains of human Iga, IgG, or IgM immunoglobulins. A total of 116 serum specimens from 58 adult patients were tested. Binding ratios between the positive and the negative serum varied between 5 and 15, occasionally being 20 or more in the IgA and IgG assays, but rarely exceeding 3 in the IgM assay. The RIA was found to be more sensitive in detecting antibodies to rotavirus than the complement fixation (CF) test, the RIA titers obtained being 50--100 times as high as the CF titers. The method described offers a possibility of evaluating the immune response to human rotavirus and of detecting recent infection.     

Studies on human lacteal rotavirus antibodies by immune electron microscopy

In vitro studies carried out by immune electron microscopy (IEM) indicate that rotavirus aggregation detected in the stools of newborn breast-fed infants with rotavirus infection is antibody-induced. Aggregation of rotavirus particles occurred with the IgA-containing fraction of expressed breast milk (EBM) obtained five days postpartum and with the IgA- and IgG-containing fractions of a pool of EBMs containing samples collected 2-3 days postpartum. Bovine milk fractions also demonstrated this activity in the IgG- and IgA-containing fraction. Studies on unfractionated EBMs from a mother who experienced a rotavirus infection during the 43rd week of lactation showed that following rotavirus infection all three major classes of rotavirus-specific antibodies were present in breast milk, this being confirmed by enzyme immunoassay.     

Oyster-associated gastroenteritis in Australia: the detection of Norwalk virus and its antibody by immune electron microscopy and radioimmunoassay

Following widespread outbreaks of oyster-associated gastroenteritis in Australia during 1978 in which Norwalk virus was implicated as the causative agent, collaborative studies were undertaken between laboratories in Australia and the United States to confirm the etiology. Immune electron microscopy (IEM) techniques were used in Australia and radioimmunoassay (RIA) methods in the United States. Norwalk virus was detected by IEM in seven of 15 faecal samples, and four were positive by RIA. A much better correlation was found with antibody determinations. Both methods demonstrated significant increases in antibody to Norwalk virus in 22 of 30 sets (73%) of "acute" and "convalescent" sera, confirming that Norwalk virus was responsible for the majority of cases. It is significant that the RIA serology was determined using Norwalk antigen originating in the United States and the IEM serology was determined using 27--30-nm particles originating in Australia.     

A study of the prevalence of rotavirus infection in children with gastroenteritis admitted to an infectious diseases hospital.

In a 12 month survey of infants and children with gastroenteritis admitted to Fairfield Hospital, Melbourne, rotavirus was found in approximately 42% of patients. This virus was detected more often during the winter months, particularly in children aged between 12 months and 3 years. Detection of rotavirus by electron microscopy was found to be more sensitive than by counterimmunoelectrophoresis. Routine bacterial and viral studies revealed that bacterial pathogens and common enteric viruses were associated with relatively few cases of gastroenteritis. There is little doubt that rotavirus is the most important aetiological agent of acute gastroenteritis in yvirus is the most important aetiological agent of acute gastroenteritis in young children in Melbourne.     

Rise per base pair in helices of double-stranded rotavirus RNA determined by electron microscopy

Regular helices of double-stranded RNA occur in nature only as the genome of certain viruses. The structure of such double-stranded RNA helices has been little studied compared to that of DNA, but some X-ray crystallographic data (Arnott, 1970; Saenger, 1984) are available. The recent advent of sequence data of bovine rotavirus RNA (Dyall-Smith et al., 1983; Elleman et al., 1983; Ward et al., 1984) has enabled us to determine by direct measurement of electron micrographs the translation, or axial distance between base pairs in RNA duplexes. Using two different spreading conditions we obtained values of 2.79 +/- 0.10 and 2.80 +/- 0.11 A. These results are consistent with the 11-fold A RNA (Arnott, 1970; Rosenberg et al., 1976) proposed for the conformation of double-stranded RNA. We included both circular and linear molecules of phi X174 RF DNA in the same preparations, and the translations for these molecules were between 3.23 +/- 0.06 and 3.29 +/- 0.05 A. Thus, double-stranded RNA contained 1.16 to 1.17 times more nucleotides per unit length than DNA.     

Solid-phase enzyme immunoassay for rotavirus antigen: faecal protease activity as a reason for false-negative results

Rabbits and guinea pigs were immunized with purified bovine rotavirus. Immunoglobulin G fractions of the resulting antisera were used in a standard four-layer solid-phase enzyme immunoassay (EIA) for rotavirus antigen in human faecal specimens. Samples negative for rotavirus in electron microscopy, when diluted in standard EIA buffers, regularly gave absorbance values lower than those obtained with buffer blank only. By further diluting of the samples the resulting absorbance values were found to increase to the blank levels. When all dilution buffers were supplemented with 1-5% of bovine serum, negative samples at any dilution gave absorbance values close to those of the buffer blanks. Similar results were obtained if the serum was replaced by 1-5 mM of phenylmethylsulphonyl fluoride, a synthetic broad spectrum serine-type protease inhibitor. Aprotinin, another protease inhibitor, was without effect. A similar inhibition pattern was obtained when faecal specimens were tested in a caseinolytic quantitative protease assay in the presence of the above inhibitors. These observations suggest that protease activity present in human faecal samples may cause false-negative results in solid-phase immunoassay for viral antigens, unless appropriate means are used to avoid this interference.     

Pseudoreplica electron microscopy for the detection of rotavirus: comparison with high-speed centrifugation electron microscopy and ELISA

Three-hundred and sixty-three stool specimens from patients with diarrhoea were examined for rotaviruses to compare the sensitivity of the pseudoreplica technique (PSD-EM) to that of high-speed centrifugation EM (HSC-EM) in relation to a commercially available (Rotazyme, Abbott) enzyme-linked immunosorbent assay (ELISA). In ELISA-positive cases, both methods were of equal sensitivity. However, in borderline (+/-) and ELISA-negative specimens, PSD-EM detected 31 of 48 (64.6%) and 18 of 229 (8%) positive specimens respectively, compared to only 22 of 48 (45.8%) and one of 229 (0.4%) positives detected by HSC-EM. PSD-EM detected a significantly higher number of positives compared to HSC-EM (p less than 0.05). In view of its simplicity, sensitivity and the fact that a relatively large number of specimens could be processed compared to HSC-EM, we consider that PSD-EM is a much better procedure for routine screening and diagnosis of viral gastroenteritis than HSC-EM.     

Enzyme-linked immunosorbent assay for the detection of human rota virus in stools

A simple method tor the detection of human rotavirus in stools is described, using a double antibody sandwich enzyme-linked immunosorbent assay. Polysterene microtitre plates were used as solid phase. Four capture antibodies were tried, bovine, egg-derived, guinea pig and monoclonal antibody to rotavirus. Both bovine and egg-derived antirotavirus labelled with horseradish peroxidase were used as the detecting antibodies. The results obtained were compared with a commercially available ELISA, Rotazyme (Abbott Laboratories), and also with the direct detection of rotavirus by electron microscopy. Bovine antibody was found to be an unsuitable capture antibody due to non-specific false positive reactions.     

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.     

A simplified microwell pseudoreplica for the detection of viruses by electron microscopy and immunoelectron microscopy

Simplified procedures for immunoelectron microscopy (IEM) and electron microscopy (EM) are described. The procedures employ the principle of agar filtration and pseudoreplication. The modification consisted of the use of microwells for storage of gels with or without antiserum (for IEM or EM, respectively) and an array of containers in which pseudoreplication and negative staining were performed. The containers were prepared from 5 ml syringes from which the needle holding parts were cut. This device enabled simultaneous and rapid handling of specimens. With Sindbis virus as a model, our microwell pseudoreplica IEM (MW-PR-IEM) was compared to six other IEM techniques and was found to be the most rapid and sensitive technique. With the MW-PR-IEM technique, the specific minimal detection limit (detection of clumps) was 1.5 x 10(7) virus particles per ml, and the non-specific detection limit (detection of single virions) was 1.8 x 10(6) virus particles per ml.     

Human rotavirus infection in infants and young children with intussusception.

Human rotavirus was detected by electron microscopy in 11 of 30 infants and young children with intussusception (37% of subjects under study). Serologic complement fixation tests revealed evidence of infection with the rotavirus in 70% of the patients examined who eliminated the rotavirus in their stools. These results indicate that human rotavirus, in addition to adenovirus, may be an infectious agent causing intussusception in infants and young children.     

Comparison of Rotazyme and direct electron microscopy for detection of rotavirus in human stools

A total of 115 stools were examined for Rotavirus, using direct electron microscopy (EM) and Rotazyme. The overall agreement was 88.7%. Of the negative results, there was 91.95% agreement. Rotazyme reactions of three-plus or more gave a 100% agreement with EM. The Rotazyme test is a useful diagnostic aid in laboratories not capable of performing EM.     

Solid-phase microtiter radioimmunoassay for detection of the Norwalk strain of acute nonbacterial,epidemic gastroenteritis virus and its antibodies

The development of microtiter solid-phase radioimmunoassays for the detection of Norwalk antigen and its antibody is described. The tests are simple to perform and are sensitive and specific. The test for antigen can be used on crude stool filtrates and suspensions. Both tests are at least as sensitive as immune electron microscopy and more sensitive than immune adherence assay.     

Comparison of electron microscopy, enzyme-linked immunosorbent assay, solid-phase radioimmunoassay, and indirect immunofluorescence for detection of human rotavirus antigen in faeces.

Four techniques were compared for their practicability, speed, and sensitivity for the detection of human rotavirus. Radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) were found to be the most sensitive means of identifying rotavirus, and, once processed, up to 40 specimens could be examined daily. Electron microscopy, although less sensitive than these techniques, had the advantage of being able to detect other viral agents present in faecal extracts. Indirect immunofluorescence failed to detect rotavirus as often as the other three methods. In laboratories where routine examination of faecal specimens from patients with gastroenteritis is required, ELISA and RIA are useful alternatives to electron microscopy.     

Development of serum and intestinal antibody response to rotavirus after naturally acquired rotavirus infection in man

The temporal characteristics of the response of rotavirus specific IgM, IgG, IgA in serum and secretory antibody in feces to rotavirus were studied in 77 hospitalized patients with rotavirus induced gastroenteritis. The response in serum was characterized by the sequential appearance of rotavirus specific IgM, IgG, and IgA antibody. The IgM antibody appeared to be higher in the acute phase of the disease and was subsequently replaced by the IgG and IgA antibodies. However, the titers of IgG rotavirus antibody in convalescent specimens of serum were found to be statistically significantly lower in patients with severe or prolonged rotavirus infection than in specimens from subjects with mild or moderate disease. Most fecal specimens collected during both the acute and convalescent phase of illness contained virus specific secretory IgA. Higher concentrations of antibody were measured in convalescent samples from patients with prolonged diarrhea and virus shedding. These observations suggest a possible relationship between the severity of rotavirus infection and the nature of systemic and secretory antibody response.     

Molecular and immunohistochemical detection of rotavirus in urinary sediment cells of children with rotavirus gastroenteritis

This is the first report showing that rotavirus infects the urinary sediment cells in immunocompetent children with rotavirus gastroenteritis. We found that inclusion-bearing cells were frequently detected in the urine samples of patients with rotavirus gastroenteritis. These cells were positive for cytokeratin, which was sometimes coexpressed with rotavirus antigen, in our immunohistochemical analysis. Moreover, in nested RT-PCR experiments, we detected rotavirus double-stranded RNA in some urine samples of patients with rotavirus gastroenteritis. We concluded that rotavirus could lead to infection of the urinary sediment cells concomitantly with rotavirus gastroenteritis.     

A simple immunofluorescent technique for the detection of human rotavirus.

If trypsin is incorporated in the tissue culture medium it is possible to carry out a sensitive immunofluorescence assay for the presence of human rotavirus. The enhanced effect of trypsin is negated by serum. It has also been established that naturally occurring enzymes in faeces enable some virus to penetrate tissue culture cells. The role of these naturally occurring enzymes in the pathogenesis of rotavirus infection is discussed.