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During the investigation of an outbreak of diarrhea in suckling rats, a virus morphologically identical to but antigenically distinct from rotaviruses was identified. The disease was characterized clinically by erythema and cracking and bleeding of the perianal skin associated with the excretion of poorly formed fecal pellets, liquid, and gas. Light microscopy-observable changes consisted of small intestinal villous atrophy, villous epithelial necrosis, and villous epithelial syncytial cell formation. The cytoplasm of the epithelial syncytial cells contained large numbers of 80-nm viral particles that were often associated with reticular aggregates of electron-dense material. Viral infection principally involved the luminal one-fourth to one-third of the intestinal villi as determined by indirect immunofluorescence. This rotavirus-like agent contained 11 double-stranded RNA segments; however, the migration pattern of these segments in polyacrylamide gels differed from the electrophoretic pattern which is characteristic of the typical rotaviruses. The agent had a buoyant density in CsCl of 1.36 to 1.4 g/cm3 and was labile at pH 3 and at 56 degrees C; however, infectivity of viral inocula was not altered by extensive treatment with ether or by pH 5 buffers. This disease, which we have named infectious diarrhea of infant rats, is the first recognized viral diarrhea of rats and appears to be a good model for the study of the recently recognized group of atypical rotaviruses.

Adult diarrhea rotavirus (ADRV) is a newly identified strain of noncultivable human group B rotavirus that has been epidemic in the People's Republic of China since 1982. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western (immuno-) blot analysis to examine the viral proteins present in the outer and inner capsids of ADRV and compared these with the proteins of a group A rotavirus, SA11. EDTA treatment of double-shelled virions removed the outer capsid and resulted in the loss of three polypeptides of 64, 61, and 41, kilodaltons (kDa). Endo-beta-N-acetylglucosaminidase H digestion of double-shelled virions identified the 41-kDa polypeptide as a glycoprotein. CaCl2 treatment of single-shelled particles removed the inner capsid and resulted in the loss of one polypeptide with a molecular mass of 47 kDa. The remaining core particle had two major structural proteins of 136 and 113 kDa. All of the proteins visualized on sodium dodecyl sulfate-polyacrylamide gel electrophoresis were antigenic by Western blot analysis when probed with convalescent-phase human and animal antisera. A 47-kDa polypeptide was most abundant and was strongly immunoreactive with human sera, animal sera raised against ADRV and against other group B animal rotaviruses (infectious diarrhea of infant rat virus, bovine and porcine group B rotavirus, and bovine enteric syncytial virus) and a monoclonal antibody prepared against infectious diarrhea of infant rat virus. This 47-kDa inner capsid polypeptide contains a common group B antigen and is similar to the VP6 of the group A rotaviruses. Human convalescent-phase sera also responded to a 41-kDa polypeptide of the outer capsid that seems similar to the VP7 of group A rotavirus. Other polypeptides have been given tentative designations on the basis of similarities to the control preparation of SA11, including a 136-kDa polypeptide designated VP1, a 113-kDa polypeptide designated VP2, 64- and 61-kDa polypeptides designated VP5 and VP5a, and several proteins in the 110- to 72-kDa range that may be VP3, VP4, or related proteins. The lack of cross-reactivity on Western blots between antisera to group A versus group B rotaviruses confirmed that these viruses are antigenically quite distinct.

Cloned cDNA copies were synthesized from the genomic RNA of the IDIR strain of group B rotavirus (GBR) isolated in Baltimore, Md. These clones were screened for hybridization with heterologous GBR to identify cDNA for use in dot hybridization experiments. In multiple screening experiments, cDNA clones derived from gene segment 3 provided the most intense hybridization signals. 32P-labeled probes were produced from one of the gene 3 clones, and these were employed in dot hybridization assays. Purified preparations of bovine GBR were detected in concentrations of greater than or equal to 0.5 ng, and GBR was detected in fecal specimens obtained from five of six infected calves. Four of six human fecal specimens containing the Baltimore strain of GBR were also positive in the hybridization assay, while GBR was identified in only one of the six specimens by means of immunoelectron microscopy. A fecal specimen obtained from a patient infected with the adult diarrhea rotavirus strain of GBR was also positive in the dot hybridization assay. Fecal specimens from uninfected humans, calves, and rats, as well as specimens containing group A rotaviruses, did not hybridize with the cloned cDNA probe.

An enzyme immunoassay inhibition assay was developed to detect rat rotavirus-like agent (RVLA) or antigenically related viruses in intestinal washings and homogenates obtained from diarrheic rats and in fecal specimens obtained from diarrheic humans. In this assay, RVLA antigens in a test sample inhibited the binding of a biotin-labeled anti-rat RVLA antibody preparation to rat RVLA antigens bound to the solid phase. Intestinal washings and homogenates obtained 1 day after RVLA infection of suckling rats inhibited the binding of the biotin-labeled antibody to the solid-phase rat RVLA antigens by 76 to 100%. The inhibition was blocked by RVLA immune rat serum but not by nonimmune rat serum. Of 27 fecal specimens obtained from diarrheic humans, 6 produced disease characteristic of rat RVLA infection when inoculated into suckling rats. Four of these six specimens produced greater than 50% inhibition in the enzyme immunoassay. Fecal specimens obtained from diarrheic humans that were determined to be negative for RVLA produced an average inhibition of 9.2%. This enzyme immunoassay appears to be a useful diagnostic and research tool for the study of infections with at least one of the antigenically distinct rotaviruses.

Non-group-A rotaviruses have been implicated with increasing frequency as causes of acute gastroenteritis in humans and other animals. However, the incidence and significance of infection with these agents, as well as appropriate diagnostic strategies for making these determinations, are largely unknown. Studies to make these determinations could be more accurately conducted if the relationship between the viral replication kinetics and the particular diagnostic method used is understood. We thus utilized the murine model of group B rotavirus infection to establish the viral replication kinetics by a variety of commonly used diagnostic methods. Enzyme immunoassay, routine negative-stain electron microscopy, solid-phase immunosorbent electron microscopy, polyacrylamide gel electrophoresis, and a dot hybridization assay were used in these studies. By enzyme immunoassay, 100% of experimentally infected suckling rats tested positive for group B rotaviral antigens at 1, 4, and 5 days postinoculation. However, only 70 and 20% of infected animals tested positive at days 2 and 3 postinoculation, respectively. Dot hybridization with a complementary DNA probe also suggested a biphasic pattern of viral antigen excretion. Evidence of the virus causing infectious diarrhea in infant rats was found only on day 1 postinoculation in samples examined by routine negative-stain electron microscopy and by polyacrylamide gel electrophoresis. Rotaviruslike particles were observed by solid-phase immunosorbent electron microscopy on days 1, 2, and 4 after viral inoculation suckling rats but were clearly the most numerous on day 1. Additionally, the enzyme immunoassay was used to quantitate the kinetics of group B rotaviral replication in the intestines of the experimentally infected animals. Levels of murine group B rotaviral antigens in intestinal samples peaked on days 1 and 4 postinoculation; however, only peak 1 represented actual intraepithelial replication of the virus. These studies thus indicate that early sample collection and selection of the appropriate diagnostic method are critical if the incidence and significance of group B and possibly other non-group-A rotaviral infections are to be accurately assessed.

We evaluated the performance characteristics of three commercially available immunoassays for the detection of rotavirus antigens in stool samples obtained from infants during the course of rotavirus gastroenteritis. Two of the assays, Bio-EnzaBead (Litton Bionetics, Charleston, S.C.) and Rotazyme (Abbott Laboratories, North Chicago, Ill.), are enzyme immunoassays, while the third, Rotalex (Medical Technology Corporation, Somerset, N.J.), is a latex agglutination assay. We tested a total of 122 samples obtained from 26 children with gastroenteritis; 56 samples, obtained from 21 children, were found to contain rotavirus antigen by a reference microplate enzyme immunoassay. Rotavirus antigen was found by the Bio-EnzaBead, Rotazyme, and Rotalex assays in 53, 42, and 29 samples, respectively. The true positivity of samples which were positive by the reference microplate assay but negative by the other assays was confirmed by a specific neutralization assay or by the visualization of bands of double-stranded RNA by polyacrylamide gel electrophoresis or both. No false-positive assay results were noted with any of the commercial assays. The sensitivity of the assays was determined to a great extent by the time after the onset of illness at which the specimen was collected. Thus, the sensitivity of commercial assays with specimens collected early in the course of illness did not differ significantly from that of the microplate assay. However, significantly lower degrees of sensitivity were noted later in the course of illness. Results of our studies indicate that all three commercial assays can accurately detect rotavirus in stools from children with rotavirus gastroenteritis. However, the choice of assay systems for use in the clinical laboratory will depend on the conditions in which stool specimens are collected and tested in the laboratory.

Fastidious viruses are generally detected in human body fluids by means of immunoassay or nucleic acid hybridization systems. These approaches can be difficult to apply to the detection of viruses which display variations in antigenic or genetic composition. Rotaviruses are examples of viruses which can display such variations. Recently identified antigenic variants, designated as non-group A rotaviruses, cannot be detected by immunoassays or nucleic acid hybridization assays which utilize reagents directed at group A rotavirus strains. The incomplete understanding of the extent of antigenic and genetic variation has inhibited the development of assay systems for all of the non-group A rotaviruses and has limited the study of their role in human disease. While rotaviruses display genetic variation, they all contain a genome which consists of double-stranded RNA. We utilized a monoclonal antibody to devise a sensitive assay for the measurement of double-stranded RNA and applied it to the detection of a wide range of rotaviruses. We found that the assay could detect double-stranded RNA from as few as 10 PFU of standard strains of group A rotaviruses. The assay system was also capable of detecting double-stranded RNA from several strains of group B rotaviruses isolated from calves, rats, and pigs at levels below those at which viral RNA could be visualized by means of polyacrylamide gel electrophoresis. When applied to the detection of double-stranded RNA in serial stools shed by rotavirus-infected children, the assay system was capable of detecting double-stranded RNA in samples in which antigen could not be detected by immunoassay. The specific nature of the double-stranded RNA detected by this assay system could be determined by the elution of the nucleic acids from the monoclonal antibody and the reaction of the RNA with specific nucleotide probes. The measurement of double-stranded RNA offers a potential method for the sensitive detection of a wide range of rotaviruses and other members of the family Reoviridae.

Group B rotaviruses (GBRs) are fastidious agents which cause enteric disease in humans and a number of other animal species. Detailed study of the role of GBRs in human disease has been hampered by the lack of immunoreagents suitable for large-scale studies. We developed a monoclonal antibody which recognizes a group-reactive antigen contained in a number of strains of GBRs. When utilized in conjunction with a hyperimmune guinea pig antiserum to GBR, this monoclonal antibody can be used in an enzyme immunoassay system to detect a wide range of GBRs. Alternatively, this monoclonal antibody can be combined with sera obtained from GBR-infected animals to devise assays which are largely specific for the homologous strain of GBR. Reactivity was not noted in either system with strains of group A or group C rotaviruses or with other members of the family Reoviridae. These results indicate that GBRs contain both group-reactive and species-specific antigens which are distinct from those found in group A rotaviruses. The availability of well-defined immunoreagents will facilitate detailed studies of GBR infections in humans and animals.

We investigated the abilities of homologous and heterologous antibody preparations to neutralize murine rotavirus in a mouse model of rotavirus infection. We found that incubation of virus with murine sera obtained from animals experimentally infected with the homologous epizootic diarrhea of infant mice strain of rotavirus resulted in inability of the virus to cause symptomatic disease in infant mice. Sera from nonimmune mice and mice infected or immunized with a heterotypic strain of rotavirus did not effectively neutralize virus in this system. On the other hand, neutralization was noted after incubation of virus with a number of immunoglobulins from other animal and human sources. The neutralizing activity of the preparations in the murine model correlated partially, but not completely, with the level of in vitro neutralizing antibody to the murine strain of rotavirus measured in a tissue culture system. In most cases, asymptomatic infection after feeding of the virus and the antibody preparation resulted in subsequent generation of an active immune response in infected animals.