Detection of rotavirus in faeces by latex agglutination.

Human rotavirus (HRV) in faeces of patients may be readily detected with high sensitivity and specificity using latex agglutination (LA) on a glass slide by making use of the cross-reactivity of anti-calf rotavirus (CRV) antibody. Latex particles were coated with anti-CRV immunoglobin. The antibody coated particles (AC-L) are specifically agglutinated by both CRV and HRV, and the agglutination is evident macroscopically within a minute. To examine the sensitivity and reliability of the LA method compared to other methods, HRV in faecal extracts of 48 infants with acute gastroenteritis was sought by the LA, reversed passive haemagglutination (RPHA) and electron microscope (EM) methods. Samples positive by the EM method were all positive by the LA method, and samples negative by EM were all negative by LA. The LA method is suitable for application as a simple clinical diagnostic test.

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.

Rapid detection of enteric adenovirus and rotavirus: a simple method using polyacrylamide gel electrophoresis.

A simple and rapid procedure for identifying adenovirus and rotavirus in stool extracts has been developed. The technique is based on polyacrylamide gel electrophoresis of the virus nucleic acid, but sample preparation is straightforward and does not entail phenol extraction or the use of a radioactive label. Furthermore, processing is not influenced by the amount of specimen obtained and is thus suitable for application as a batch testing method. This approach removes the need for specific antisera, which are not readily available since most of these viruses cannot be grown using routine tissue culture procedures. Trials in this laboratory have indicated that the technique is of comparable sensitivity to electron microscopy.

Evaluation of a commercial enzyme immunoassay kit for rotavirus detection.

A commercial enzyme immunoassay kit (Rotazyme) was compared to electron microscopy for the detection of rotavirus in stool specimens collected during diarrhea outbreaks at day care centers in Houston. EIA was more sensitive than EM and detected SA-11 rotavirus which titered 2.0 X 10(3) PFU/ml.

A rapid and sensitive solution hybridisation assay for the quantitative determination of specific viral RNA sequences.

A solution hybridisation assay has been developed which allows the quantitation of specific viral (RNA) sequences in infected cells. The assay makes use of single-stranded (ss) RNA probes of known polarity synthesised at high specific activity in vitro from cDNA clones of the relevant viral gene by the SP6 or T7 RNA polymerase. These probes are used together with samples containing the RNA to be detected at a known concentration to construct a calibration curve to relate RNase resistant radioactivity following solution hybridisation to amount of RNA. The amount of RNA in experimental samples is then determined using the calibration curve that is produced each time the assay is performed. The UKtc strain of Rotavirus growing in BSC-1 cells was used to develop this method but with the substitution of suitable cDNA clones it could be applied to any viral system.

The use of markers in immune electron microscopy.

Immune electron microscopy (IEM) cannot be used successfully for structures that do not have recognisable morphology. However, at least some of these structures or components are related antigenically to recognisable antigens or viruses. We have therefore mixed unknown antigens with known markers and looked for the presence of mixed aggregates. The present study examined a low molecular weight subunit of rotavirus and a micellar form of hepatitis B surface antigen. In both cases mixed immune aggregates were found showing that the unknown components had antigens in common with the established virus or antigen.

The cultivation of human rotavirus, strain 'Wa', to high titer in cell culture and characterization of the viral structural polypeptides.

The structural proteins of the 'Wa' (serotype 2) strain of human rotavirus have not been described previously. Single-cycle virus growth in MA-104 cells using 5 micrograms/ml of trypsin in the growth medium was rapid with maximal viral yields (approximately 10(6) PFU/ml) obtained 10-12 h post-infection. There was a continuous progression of cytopathic effect (CPE) from 6- to 5-h post-infection. Under conditions of multiple-cycle growth, a greater concentration of trypsin (40 micrograms/ml) in the growth medium was required to obtain rapid progression of CPE and production of a high titer (approximately 10(7) PFU/ml) of infectious (double-shelled) virus. Single- and double-shelled virions were separated by isopycnic centrifugation in CsCl and analyzed by SDS-PAGE. Five proteins with molecular weights of 116,000, 92,000, 88,000, 84,000 and 41,000 were identified as components of the inner shell and four proteins with molecular weights of 60,000, 38,000, 32,000 and 27,000 were located in the outer shell.

An assessment of the sensitivity of three methods for the detection of rotavirus.

A comparison was made of the sensitivity of a current commercial ELISA for detecting rotavirus in faecal specimens with the more complex, technically demanding systems of electron microscopy and polyacrylamide gel electrophoresis. Even after modification, the ELISA failed to detect 22% of specimens with particles identifiable by electron microscopy. Polyacrylamide gel electrophoresis failed to identify 2 out of 50 specimens with particles present but did distinguish 2 group C rotaviruses.

Shifts in the electrophoretic pattern on the RNA genome of rotaviruses under different electrophoretic conditions.

Substantial differences in the RNA electrophoretic patterns of rotaviruses were observed when the conditions for gel electrophoresis were varied. Electropherotypes which seem alike under one set of conditions, may appear significantly different under other conditions such as distinct proportions of acrylamide and bisacrylamide or different running buffer. Different patterns were observed for the same rotavirus RNA sample even when different voltages were applied to otherwise identical runs; these differences were probably caused by the increasing amount of heat generated at higher voltage. Although the conditions of electrophoresis did not have as pronounced an effect on 'double-stranded' as on 'single-stranded' RNA, precautions should be taken to obtain comparable and reproducible results. A standard methodology for 'electropherotyping' of rotaviruses is therefore suggested.

A rapid screening assay for detecting individual RNA species in field isolates of rotaviruses.

A method is described that allows the rapid screening of field isolates of rotavirus for the detection of specific viral RNA segments. Cloned cDNA copies of viral genomic RNAs are employed for detection in the assay which makes use of the dot-hybridization technique of Thomas (1980). The assay developed was shown to be sufficiently sensitive and specific to allow the genetic composition of virions to be determined over a wide range of concentrations. The feasibility of using the method developed for both the analysis of genetic reassortants prepared in vitro and for screening field isolates to detect putative genome reassortment events is demonstrated.

Characterization of rotaviruses isolated from pigs with diarrhoea in Venezuela.

The prevalence of porcine rotavirus infection was studied in 15 different herds located in the north-western region of Venezuela. The presence of rotavirus was studied by direct electron microscopy (EM) and by an enzyme-linked immunosorbent assay (ELISA). From 136 samples analyzed during the six months of the study (September 1983-February 1984), 38 (27.9%) were found to be positive for rotaviruses, with infection more common in animals that were 4-6 weeks old. Atypical rotaviruses were not detected in any of the samples examined. Most rotavirus positive specimens were subgrouped using specific monoclonal antibodies in an ELISA test. The majority of the samples (26 out of 38) were found to exhibit Subgroup I antigenicity. Only two specimens, collected from the same herd in two consecutive months, were found to belong to Subgroup II. To characterize further the circulating rotaviruses, electrophoretic analysis of the RNA genome was performed on samples selected from nine different herds. Great variability in the RNA electropherotypes was observed. No correlation was found between subgroup specificity and the migration of the two smaller segments (Genes 10 and 11), as has been described for human rotaviruses.

Genomic variation among avian rotavirus-like viruses detected by polyacrylamide gel electrophoresis.

The genomes of five turkey and two chicken rotavirus-like virus (RVLV) strains were compared with the genome of a reference turkey RVLV strain by co-electrophoresis in polyacrylamide gels. The genomes of these avian RVLV strains could be distinguished from the reference strain genome by differences in the mobilities of from three to 10 segments.

Electropherotypic analysis of rotaviruses isolated from turkeys.

From 1980 to 1984, several flocks of turkeys in Minnesota exhibiting signs of clinical enteritis were examined for viruses. Electron microscopic (EM) examination of fecal specimens from 35 flocks revealed the presence of rotavirus particles. Rotaviruses were successfully isolated in cell cultures from only 24 of these positive fecal specimens. Double-stranded RNA (dsRNA) preparations made from these 24 cell-culture isolates and from the remaining 11 fecal samples that were rotavirus-positive on EM examination were analyzed by polyacrylamide gel electrophoresis for genetic differences in their genomes. The study revealed eight distinct electropherotypes among the rotavirus dsRNA preparations. Atypical dsRNA migration patterns were recognized only in preparations of dsRNA from fecal materials.

Physical, chemical, and serological characterization of avian rotaviruses.

Physical, chemical, and serological characterization of rotavirus isolates from turkeys was done. Cesium chloride (CsCl)-gradient isopycnic centrifugation of infected cell cultures revealed the presence of rotavirus particles of three different densities. They were double-shelled, single-shelled, and core particles. The double-shelled particles had a buoyant density (in CsCl) of 1.34 g/cml3, and that of single-shelled particles in CsCl was 1.36 g/cm3. The buoyant density of core particles in CsCl was greater than 1.40 g/cm3. These rotavirus isolates were not inactivated by chloroform and were relatively stable at pH 3.0. Their replication was not affected by 5-bromo-2'-deoxyuridine. Avian rotaviruses were not completely inactivated by heat treatment of 56 C for 8 hr. All six avian rotavirus isolates examined were antigenically related to each other. However, there was no antigenic relationship between mammalian rotaviruses and the avian rotavirus isolates examined.

Primary isolation and identification of avian rotaviruses from turkeys exhibiting signs of clinical enteritis in a continuous MA 104 cell line.

Avian rotaviruses were isolated from turkeys with enteritis using MA 104 cell line. MA 104 cells were suitable for primary isolation and propagation of avian rotaviruses. Trypsin appeared essential for the enhancement of infectivity and the occurrence of cytopathic effect (CPE). Serum neutralization (SN), electron microscopy (EM), and analysis of genomic RNA were done to identify and confirm the identity of rotaviruses. Electrophoretic migration patterns of genomic RNA from avian rotaviruses were examined, and they were compared with those from mammalian rotaviruses. The migration patterns differed between these groups.

Subgroup classification of porcine group-A rotaviruses, using monoclonal antibodies in an enzyme-linked immunosorbent assay.

Fifty-six samples of feces and intestinal contents from nonvaccinated diarrheal pigs with rotavirus infections were tested, using a subgroup (SGP)-specific ELISA, to determine rotavirus SGP classification. Forty-one percent (23/56) were SGP 1, 25% (14/56) were SGP 2, and 34% (19/56) were not classifiable. For classifiable samples, the geographic distribution for SGP 1 and SGP 2, respectively was: 60%/40% from Ohio (n = 15), 63%/37% from other midwestern states (Iowa, Minnesota, Nebraska, South Dakota; n = 16), and 67%/33% from Canada (n = 6). Thirty-seven SGP-classifiable samples were categorized according to age of pigs. Of pigs less than or equal to 1 week old, 22% of samples were SGP 1 (n = 8), and 14% (n = 5) were SGP 2. Of samples from 1- to 2-week-old pigs, 8% were SGP 1 (n = 3), and 5% were SGP 2 (n = 2). Of samples from 2- to 3-week-old pigs, 5% were SGP 1 (n = 2), and 8% were SGP 2 (n = 3). Of samples from 3- to 4-week-old pigs, 5% were SGP 1 (n = 2), and 3% were SGP 2 (n = 1). Of samples from pigs greater than 4 weeks old, 22% were SGP 1 (n = 8) and 8% were SGP 2 (n = 3). Double-stranded RNA extracted from positive controls and from 10 selected field samples (5 from SGP 1 and 5 from SGP 2) was electrophoresed in polyacrylamide gels to detect correlation between subgroup classification by ELISA and long or short double-stranded RNA electrophoretic-migration patterns. All SGP-1 and -2 rotavirus samples tested had typical long double-stranded RNA electrophoretic-migration patterns.

Classification of rotaviruses: report from the World Health Organization/Food and Agriculture Organization Comparative Virology Program.

The Reoviridae working team was established under the World Health Organization/Food and Agriculture Organization Comparative Virology Program in 1975. The generic name rotavirus has been adopted for the reovirus-like agents associated with diarrhea in man and animals, and the Nebraska calf diarrheal virus strain of bovine rotavirus has been selected as a candidate reference virus. Stocks of this virus and of gnotobiotic calf antiserum have been prepared. Antigenic differences among rotaviruses isolated from different species were recognized on the basis of virus-neutralization tests; a possible association between antigen specificity and variation in the RNA segments and structural proteins of rotaviruses was noticed.

[Enzyme immunoassay for rotavirus using nylon as the solid phase]. / Enzimoinmunoanálisis para rotavirus con nylon como fase sólida.

An enzyme-linked immunoassay (EIA) to detect Rotavirus in stools is described. Antibodies prepared in rabbits were immobilized on small nylon cubes as capture phase and enzyme conjugated antibodies were used to reveal the reaction. The conjugate was prepared with horseradish peroxidase by the Nakane periodate oxidation method. The solid phase consisted of 3 mm nylon cubes (66 CNL Du-cilo) previously submitted to partial acid hydrolysis to liberate amino-reactive groups. Glutaraldehyde was employed to couple the capturing antibody to the solid phase resulting in a covalent linkage between the gamma-globulin and the nylon. Phenylenediamine in citrate buffer pH 5.0 with 0.5% hydrogen peroxide was used as revealing substrate. EIA was performed as follows: stools watery extracts were incubated 1 h at 37 degrees C with antibody-treated nylon cubes, and then with enzyme conjugate, rinsed with distilled water and substrate-added. Samples developing colour, with optical density of at least 0.350 at 492 nm, were considered positive. The method showed good correlation with a commercial kit.

[Rapid diagnosis of rotavirus infections by RNA electrophoresis on polyacrylamide gel]. / Bystraia diagnostika rotavirusnykh infektsii putem élektroforeza RNK v poliakrilamidnom gele.

A method of rapid diagnosis of rotavirus infections is described. It is based on the isolation of viral RNA from the feces of patients with viral gastroenteritis and determination of RNA mobility by 6% polyacrylamide gel electrophoresis. It was demonstrated that rotavirus infection could be diagnosed after long-term storage of fecal specimens as well. The method is of interest for epidemiological and genetic studies of human rotavirus diseases.

[The RNA electrophoretypes of the rotaviruses circulating in Moscow and Leningrad in the winter of 1987-1988]. / Elektroforetipy RNK rotavirusov, tsirkuliruiushchikh v Moskve i Leningrade zimoi 1987--1988 gg.

Analysis of electrophoretypes of RNA of rotavirus which had circulated in Moscow and Leningrad in the winter of 1987-1988, detected by enzyme immunoassay (EIA), was carried out. RNA electrophoresis was performed in 10% polyacrylamide gel (PAG) followed by silver staining, Most of the strains isolated in Moscow and Leningrad had a long phoretype (67% and 77%, respectively. The greatest variations in PAG mobility were found in segments 2, 3, and 7-9, segments 1, 4, 10, and 11 showed most unchangeable mobility. According to the pattern of segment migration, 3 variants of the long phoretype and 5 variants of the short phoretype were distinguished. In Moscow the ist variant (confluent 2nd and 3rd segments as well as 7th and 8th segments) of the long phoretype was predominant, which was isolated in approximately 70% of all cases of infection; in Leningrad the dominating variant was intermediate between the 1st and 2nd phoretype (slower migration of the 2nd segment). Variants of the sport phoretype were characterized by greater variability and lack of the dominating strain. The potentials of rotavirus RNA electrophoresis as a method of molecular epidemiology are discussed.