The polypeptide compositions of single-shelled and double-shelled simian rotavirus particles were modified by exposure to proteolytic enzymes. Specificially, a major outer capsid polypeptide (VP3) having a molecular weight of 88,000 in double-shelled particles was cleaved by trypsin to yield two polypeptides, VP5* and VP8* (molecular weights, 60,000 and 28,000, respectively). The cleavage of VP3 by enzymes that enhanced infectivity (trypsin, elastase, and pancreatin) yielded different products compared to those detected when VP3 was cleaved by chymotrypsin, which did not enhance infectivity. The appearance of VP5* was correlated with an enhancement of infectivity. Cleavages of the major internal capsid polypeptide VP2 were also observed. The VP2 cleavage products had molecular weights similar to those ofknown structural and nonstructural rotavirus polypeptides. We confirmed the precursor-product relationships by comparing the peptide maps of the polypeptides generated by digestions with V-8 protease and chymotrypsin. The remaining rotavirus structural polypeptides, including the outer capsid glycoproteins (VP7 and 7a), were not altered by exposure to pancreatic enzymes. Cleavage of VP3 was not required for virus assembly, and specific cleavage of the polypeptides occurred only on assembled particles. We also discuss the role of cleavage activation in other virus-specific biological functions (e.g., hemagglutination and virulence).

Rotavirus gene products were examined, with the simian rotavirus SAll as a model. The endogenous viral RNA-dependent RNA polymerase associated with single-shelled virus particles or with activated double-shelled particles was used to synthesize viral RNA transcripts. Sedimentation velocity sucrose gradient analysis of the RNA transcripts revealed four peaks at 9S, 12S, 14S, and 18S, whereas agarose gel electrophoresis under partially denaturing conditions revealed eight groups of RNA species ranging in molecular weight from 2 x 105 to 1.2 x 106. The transcripts synthesized in vitro were active in an mRNA-dependent cell-free translation system derived from rabbit reticulocytes. The transcripts directed the synthesis of 11 polypeptides that had molecular weights ranging from 125,000 to 20,000 when analyzed by electrophoresis in sodium dodecyl sulfatepolyacrylamide gels. The products of in vitro translation were compared with polypeptides from purified virus and those synthesized in infected cells. Several of the polypeptides synthesized in vitro were designated as structural polypeptides by comparing the molecular weights determined by polyacrylamide gel electrophoresis analysis or by precipitation with hyperimmune serum prepared against purified virus. Three of the viral structural polypeptides (VP4,-5, and-5a) were not synthesized in vitro as primary gene products, demonstrating that processing must occur for the production of some structural polypeptides. Other in vitrosynthesized polypeptides were tentatively identified as either precursors to the viral glycoproteins or nonstructural polypeptides.

Antigenically distinct rotaviruses, i.e., viruses morphologically identical to conventional rotaviruses by electron microscopy, yet lacking the common group antigen(s) detected by an enzyme-linked immunosorbent assay, were found in 2 of 51 fecal samples from Bulgarian infants with rotavirus gastroenteritis. These antigenically distinct viruses contained 11 segments of double-stranded RNA, but they demonstrated a unique RNA migration profile after electrophoresis of the genome RNA in polyacrylamide gels. This report confirms the presence of a new group of rotaviruses in humans. The significance of these viruses is currently unknown, and specific diagnostic tests must be developed for epidemiological studies to determine their role as human and veterinary pathogens and to evaluate their impact on proposed vaccine development programs.

We used DNA-DNA hybridization to characterize a mycobacterial isolate, strain Linda, that was obtained from a patient with Crohn's disease and that has been reported to cause ileitis in experimental animals. We also investigated the association of this mycobacterium with Crohn's disease. Our results identified Mycobacterium strain Linda as a strain of Mycobacterium paratuberculosis, the etiologic agent of Johne's disease, a disease of ruminants that has some superficial resemblance to Crohn's disease. Sequences that hybridized with strain Linda DNA probes were detected in DNA extracted from human intestinal tissues from patients with Crohn's disease, ulcerative colitis, and noninflammatory bowel disease. These hybridizing DNA sequences were more prevalent in the muscle layers than in the intestinal mucosa, making it unlikely that they represented DNA from bacterial contaminants in the intestinal lumen. Measurement of the melting temperatures of the DNA-DNA hybrids formed between strain Linda probes and tissue DNAs indicated that the related sequences detected were of mycobacterial origin but were not identical to each other or to strain Linda DNA. These results do not support the proposed specific relationship between Mycobacterium strain Linda and Crohn's disease. The possible etiologic role of mycobacteria in Crohn's disease is discussed.

Group B rotaviruses have been responsible for annual epidemics of severe diarrhea affecting both adults and children in China. We developed a specific and sensitive enzyme-linked immunosorbent blocking assay to detect antibody to group B rotaviruses that will be useful to assess the role of group B rotavirus infections as a cause of human gastroenteritis. We tested 219 human sera and 18 immunoglobulin pools collected from eight countries for antibodies to both group A and group B rotaviruses. Overall, a low proportion (10 of 237 or 4.2%) of sera contained antibody to group B rotaviruses. Antibody to group B rotavirus was detected in only 1 of 155 serum samples from healthy or hospitalized individuals in the United States, including patients with the chronic inflammatory bowel diseases Crohn's disease and ulcerative colitis. No antibody was detected in 15 serum samples from Australia and from an outbreak of gastroenteritis on a cruise ship or in nine immunoglobulin pools from Japan and the United Kingdom. Antibody to group B rotaviruses was detected in 8 convalescent-(but not acute-)phase serum samples from Chinese patients with group B gastroenteritis, in five immunoglobulin pools from China, in 1 of 6 serum samples from Chinese students in the United States, and in 1 each of 10 serum samples from Kenya, 20 from Thailand, and 15 from Canada. In contrast, most of these samples (226 of 237 or 95.4%) had antibody to group A rotaviruses. These results indicate that human infection with group B rotavirus has not been widespread in areas outside China. Seroconversion observed between the acuteand convalescent-phase serum samples from China also suggests that infections with this virus are primary infections. Continued surveillance for this new group of rotaviruses should determine whether the many susceptible people become infected or whether other factors influence the severe pathogenicity of human infections with these viruses in China.