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PubMed1984

Improved enzyme immunoassays for the detection of antigens in fecal specimens. Investigation and correction of interfering factors.

Solid phase enzyme immunoassays (EIA) are widely used for the detection of infectious agents in body fluids such as stool specimens. However, we found that stool specimens contained substances which desorb from 50% to 68% of the immunoreactant from solid phase surfaces. This desorbing activity decreased the sensitivity of EIA systems for toxin A of C. difficile, rotavirus and adenovirus. The desorbing activity in stool specimens was partially heat labile at 56 degrees C for 30 min, was present in stool fractions corresponding to an estimated molecular weight of 25,000 and was shown to degrade solid phase protein. In addition, the desorbing activity was partially reversed by specific and nonspecific protease inhibitors. Thus, the desorption may reflect the enzymatic activity of stool proteases. The desorption was markedly reduced by diluting specimens in 50% fetal calf serum or an acid-protein buffer such as 0.25 M citrate buffer, pH 4.7, containing 5% bovine serum albumin. These diluents were shown to improve the recovery of toxin A of C. difficile, rotavirus and adenovirus in EIA systems for these antigens.

Alkaline Phosphatase↗
PubMed1999

Genetic and antigenic characterization of a serotype P[6]G9 human rotavirus strain isolated in the United States.

During an epidemiologic survey of rotavirus infections established to monitor the prevalent G serotypes circulating in the United States, human P[6]G9, subgroup I rotavirus strains causing symptomatic infections were identified as the fourth most common serotype. In this report we describe the molecular and antigenic characterization of one of these P[6]G9 isolates (US1205). Neutralization and sequencing studies have demonstrated that both outer capsid proteins, VP7 and VP4, of US1205 are closely related to but genetically and antigenically distinguishable from those of standard G9 strains (e.g., F45, WI61) and standard P2A[6] strains (e. g., ST3, M37). Thus the complete antigenic type of US1205 is P2A[6]G9, subgroup I. Sequence analysis of the VP6 and NSP4 genes of US1205 indicates that strain US1205 possessed VP6 subgroup I and NSP4A genotype specificities. Finally, Northern hybridization studies suggest that the P[6]G9 strains are closely related to members of the DS-1 genogroup except for their P[6] VP4 gene, which has been commonly identified in strains of both major human genogroups, and their G9 VP7 gene, which may have been derived by reassortment with a Wa genogroup strain. Examination of historic collections and prospective surveillance of strains will be needed to determine whether this strain has been present for some time or if it is emerging to compete with the other common serotypes of rotavirus.

Amino Acid Sequence↗
PubMed1997

Rotavirus virus-like particles administered mucosally induce protective immunity.

We have evaluated the immunogenicity and protective efficacy of rotavirus subunit vaccines administered by mucosal routes. Virus-like particles (VLPs) produced by self-assembly of individual rotavirus structural proteins coexpressed by baculovirus recombinants in insect cells were the subunit vaccine tested. We first compared the immunogenicities and protective efficacies of VLPs containing VP2 and VP6 (2/6-VLPs) and G3 2/6/7-VLPs mixed with cholera toxin and administered by oral and intranasal routes in the adult mouse model of rotavirus infection. VLPs administered orally induced serum antibody and intestinal immunoglobulin A (IgA) and IgG. The highest oral dose (100 microg) of VLPs induced protection from rotavirus challenge (> or = 50% reduction in virus shedding) in 50% of the mice. VLPs administered intranasally induced higher serum and intestinal antibody responses than VLPs administered orally. All mice receiving VLPs intranasally were protected from challenge; no virus was shed after challenge. Since there was no difference in immunogenicity or protective efficacy between 2/6- and 2/6/7-VLPs, protection was achieved without inclusion of the neutralization antigens VP7 and VP4. We also tested the immunogenicities and protective efficacies of 2/6-VLPs administered intranasally without the addition of cholera toxin. 2/6-VLPs administered intranasally without cholera toxin induced lower serum and intestinal antibody titers than 2/6-VLPs administered with cholera toxin. The highest dose (100 microg) of 2/6-VLPs administered intranasally without cholera toxin resulted in a mean reduction in shedding of 38%. When cholera toxin was added, higher levels of protection were achieved with 10-fold less immunogen. VLPs administered mucosally offer a promising, safe, nonreplicating vaccine for rotavirus.

Adjuvants, Immunologic↗
PubMed1996

Human rotavirus VP4 contains strain-specific, serotype-specific and cross-reactive neutralization sites.

The neutralization epitopes of human rotavirus VP4 were studied by using a panel of neutralization monoclonal antibodies previously shown to be strain-specific (RV-3:3), serotype-specific (RV-5:2, ST-3:3) or cross-reactive (F45:4). Antigenic variants of human rotaviruses RV-3, ST-3, RV-5 and F45 resistant to neutralization by the appropriate of VP4 specific monoclonal antibodies (RV-3:3, ST-3:3, RV-5:2 and F45:4 respectively) were selected. By nucleotide sequence analysis and single strand conformational polymorphism analysis of these variants, three sites of neutralization on VP5* and one site on VP8* were identified. At or near to the putative fusion region on VP5*, a strain-specific site (aa383), a serotype P1A-P2 cross-reactive site (aa392) and a serotype P2-specific site (aa397) were found. On VP8*, a serotype P1B-specific site at aa148 was detected. These results confirmed the importance of the putative fusion region in neutralization and have identified a new neutralization site in the hypervariable region of VP8* which is specific for serotype P1B human rotaviruses.

Amino Acid Sequence↗
PubMed2006

N- and C-terminal cooperation in rotavirus enterotoxin: novel mechanism of modulation of the properties of a multifunctional protein by a structurally and functionally overlapping conformational domain.

Rotavirus NSP4 is a multifunctional endoplasmic reticulum (ER)-resident nonstructural protein with the N terminus anchored in the ER and about 131 amino acids (aa) of the C-terminal tail (CT) oriented in the cytoplasm. Previous studies showed a peptide spanning aa 114 to 135 to induce diarrhea in newborn mouse pups with the 50% diarrheal dose approximately 100-fold higher than that for the full-length protein, suggesting a role for other regions in the protein in potentiating its diarrhea-inducing ability. In this report, employing a large number of methods and deletion and amino acid substitution mutants, we provide evidence for the cooperation between the extreme C terminus and a putative amphipathic alpha-helix located between aa 73 and 85 (AAH73-85) at the N terminus of DeltaN72, a mutant that lacked the N-terminal 72 aa of nonstructural protein 4 (NSP4) from Hg18 and SA11. Cooperation between the two termini appears to generate a unique conformational state, specifically recognized by thioflavin T, that promoted efficient multimerization of the oligomer into high-molecular-mass soluble complexes and dramatically enhanced resistance against trypsin digestion, enterotoxin activity of the diarrhea-inducing region (DIR), and double-layered particle-binding activity of the protein. Mutations in either the C terminus, AAH73-85, or the DIR resulted in severely compromised biological functions, suggesting that the properties of NSP4 are subject to modulation by a single and/or overlapping highly sensitive conformational domain that appears to encompass the entire CT. Our results provide for the first time, in the absence of a three-dimensional structure, a unique conformation-dependent mechanism for understanding the NSP4-mediated pleiotropic properties including virus virulence and morphogenesis.

Cell Line↗
PubMed1998

Effect of water-based microencapsulation on protection against EDIM rotavirus challenge in mice.

We determined the capacity of microcapsules formed by the combination of sodium alginate, an aqueous anionic polymer, and spermine hydrochloride, an aqueous cationic amine, to enhance protection against rotavirus challenge in mice. Adult BALB/c mice were orally inoculated with either free or microencapsulated rotavirus (simian rotavirus strain RRV) and challenged 6 or 16 weeks later with murine rotavirus strain EDIM. Virus-specific humoral immune responses were determined at the time of challenge and 4 days after challenge by intestinal fragment culture. We found that spermine-alginate microcapsules enhanced protection against challenge 16 weeks after immunization but not 6 weeks after immunization. Quantities of virus-specific immunoglobulin A produced by small intestinal lamina propria lymphocytes were correlated with the degree of protection against challenge afforded by spermine-alginate microcapsules. Possible mechanisms by which microcapsules enhance protection against rotavirus challenge are discussed.

Alginates↗
PubMed2000

The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6.

Rotavirus NSP5 is a non-structural phosphoprotein with putative autocatalytic kinase activity, and is present in infected cells as various isoforms having molecular masses of 26, 28 and 30-34 kDa. We have previously shown that NSP5 forms oligomers and interacts with NSP6 in yeast cells. Here we have mapped the domains of NSP5 responsible for these associations. Deletion mutants of the rotavirus YM NSP5 were constructed and assayed for their ability to interact with full-length NSP5 and NSP6 using the yeast two-hybrid assay. The homomultimerization domain was mapped to the 20 C-terminal aa of the protein, which have a predicted alpha-helical structure. A deletion mutant lacking the 10 C-terminal aa (DeltaC10) failed to multimerize both in yeast cells and in an in vitro affinity assay. When transiently expressed in MA104 cells, NSP5 became hyperphosphorylated (30-34 kDa isoforms). In contrast, the DeltaC10 mutant produced forms equivalent to the 26 and 28 kDa species, but was poorly hyperphosphorylated, suggesting that multimerization is important for this proposed activity of the protein. The interaction domain with NSP6 was found to be present in the 35 C-terminal aa of NSP5, overlapping the multimerization domain of the protein, and suggesting that NSP6 might have a regulatory role in the self-association of NSP5. NSP6 was also found to interact with wild-type NSP5, but not with its mutant DeltaC10, in cells transiently transfected with plasmids encoding these proteins, confirming the relevance of the 10 C-terminal aa for the formation of the heterocomplex.

Animals↗
PubMed1993

Sequence analysis of group B rotavirus gene 1 and definition of a rotavirus-specific sequence motif within the RNA polymerase gene.

The complete nucleic acid sequence was determined for the largest genomic segment of the IDIR strain of group B rotavirus and compared with RNA polymerase genes of rotavirus groups A and C as well as other RNA viruses. IDIR gene 1 contained 3509 bases with a single, long open reading frame which encoded a deduced polypeptide of 1159 amino acids (MW = 131.6 kDa; pl 8.851). The deduced amino acid sequence of IDIR gene 1 shared 50% similar sequences and 27.6% identical sequences with VP1 of the RF strain of group A rotavirus. IDIR gene 1 also contained 45.4% similar and 26.5% identical amino acid sequences in comparison with gene 1 of the Cowden strain of group C rotavirus. Amino acids 643-689 of IDIR gene 1 corresponded to the conserved viral RNA polymerase domains, "SG . . . T . . . NS . . N" and "GDD." Within these domains, group A, B, and C rotaviruses displayed substantial homologies that were not shared with other RNA viruses. These sequences indicated the presence of highly conserved structural or functional components among groups of rotaviruses which were otherwise quite heterogeneous. The identification of rotavirus-specific residues within RNA polymerase sequence may prove valuable in devising strategies aimed at the control of rotavirus replication and infection.

Amino Acid Sequence↗
PubMed1989

The VP7 gene of a new G serotype of human rotavirus (B37) is similar to G3 proteins in the antigenic c region.

The human rotavirus isolate B37 has a characteristic "super-short" RNA electropherotype and has been shown to represent a new VP7 serotype (M. J. Albert, L. E. Unicomb, and R. F. Bishop, 1987, J. Clin. Microbiol. 25, 183-185). The VP7 gene was cloned, and its nucleotide and predicted amino acid sequences were compared to other published VP7 gene sequences. Consistent with the serological evidence, two major antigenic regions of the B37 VP7 (i.e., regions A and B) differ in sequence from those of other G serotypes. Unexpectedly, the C antigenic region shows close similarity to G3 rotaviruses, but we were unable to detect a serological relationship using serotype 3 monoclonal antibodies.

Amino Acid Sequence↗
PubMed2003

The N- and C-terminal regions of rotavirus NSP5 are the critical determinants for the formation of viroplasm-like structures independent of NSP2.

Molecular events and the interdependence of the two rotavirus nonstructural proteins, NSP5 and NSP2, in producing viroplasm-like structures (VLS) were previously evaluated by using transient cellular coexpression of the genes for the two proteins, and VLS domains as well as the NSP2-binding region of NSP5 were mapped in the context of NSP2. Review of the previous studies led us to postulate that NSP2 binding of NSP5 may block the N terminus of NSP5 or render it inaccessible and that any similar N-terminal blockage may render NSP5 alone capable of producing VLS independent of NSP2. This possibility was addressed in this report by using two forms of NSP5-green fluorescent protein (GFP) chimeras wherein GFP is fused at either the N or the C terminus of NSP5 (GFP-NSP5 and NSP5-GFP) and evaluating their VLS-forming capability (by light and electron microscopy) and phosphorylation and multimerization potential independent of NSP2. Our results demonstrate that NSP5 alone can form VLS when the N terminus is blocked by fusion with a nonrotavirus protein (GFP-NSP5) but the C terminus is unmodified. Only GFP-NSP5 was able to undergo hyperphosphorylation and multimerization with the native form of NSP5, emphasizing the importance of an unmodified C terminus for these events. Deletion analysis of NSP5 mapped the essential signals for VLS formation to the C terminus and clearly suggested that hyperphosphorylation of NSP5 is not required for VLS formation. The present study emphasizes in general that when fusion proteins are used for functional studies, constructs that represent fusions at both the N and the C termini of the protein should be evaluated.

Animals↗
PubMed1992

Transient expression and mutational analysis of the rotavirus intracellular receptor: the C-terminal methionine residue is essential for ligand binding.

Maturation of rotavirus involves an intracellular membrane budding event in which the single-shelled icosahedral particle interacts with a virus-encoded receptor glycoprotein, NS28, that is located in the rough endoplasmic reticulum membrane. The receptor is a tetramer and is oriented with the C-terminal 131 amino acids on the cytoplasmic side of the membrane (A.R. Bellamy and G.W. Both, Adv. Virus Res. 38:1-48, 1990). We have used the T7-vaccinia virus transient expression system to deliver mutant variants of the NS28 gene to CV1 cells in order to assess the effects of site-specific modifications on receptor function. Three types of mutant proteins have been constructed by altering the extreme C-terminal methionine, cysteine residues within the third hydrophobic domain, and internal residues located within the cytoplasmic portion of the receptor, respectively. Deletion or conservative substitution of the C-terminal methionine completely abolishes receptor activity. Substitution of cysteine residues has no effect on receptor activity or on the ability of the receptor to adopt its native oligomeric state. Internal deletions result only in a reduction in the level of binding. An N-terminally truncated form of the receptor, containing only the cytoplasmic domain, retains full receptor activity and can form membrane-associated tetramers.

Animals↗
PubMed1989

Epidemiology of typical and atypical rotavirus infections in New Zealand pigs.

Polyacrylamide gel electrophoresis (PAGE) and enzyme-linked immunosorbent assay (ELISA) were employed to investigate the epidemiology of typical and atypical rotavirus infections in five piggeries. Of 152 faecal samples examined, 46 (30 per cent) were positive by ELISA for group A rotavirus. Rotaviruses with electrophoretic patterns resembling groups A, B and C were detected. At least two and up to five different rotavirus electrophoretypes (typical and/or atypical) were detected in each of the five piggeries. Out of 152 faecal samples examined, 28 (18 per cent) contained rotaviruses with group A electrophoretypes, 9 (6 per cent) with group C but only 1 with Group B. Six samples contained both group A and group C rotaviruses. No common electrophoretypes of group A or C rotaviruses were detected in these five piggeries. The PAGE technique was also used to analyze group A rotavirus isolated sequentially from another piggery over a three year period. A single electrophoretype was found during the first two years, but in the third year a different electrophoretype was detected.

Journal Article↗
PubMed1994

Reduction of virus shedding by B. bifidum in experimentally induced MRV infection. Statistical application for ELISA.

The protective effect of a human strain of Bifidobacterium bifidum (B. bifidum) against murine group A rotavirus (MRV) was examined in the intestines of BALB/c infected mice. In experiments designed to determine whether B. bifidum mediated MRV shedding during diarrheal disease, pregnant dams (and their expected litters) were randomly assigned to the following groups: (1) mice infected with MRV alone; (2) B. bifidum-treated + MRV-infected mice; (3) B. bifidum-treated controls; and (4) saline control animals. An enzyme-linked immunosorbent assay (ELISA) for the detection of group A rotavirus was used to measure virus protein. The sensitivity of the MRV antigen detector ELISA was determined by serially diluting the rotavirus antigen in test samples. Antigen was detected in dilution ranges of 1:256-1:4096 during the acute phase and 1:16-1:512 in the recovery phase of MRV clinical disease, in the samples tested. Treatment with B. bifidum significantly reduced shedding of MRV antigen (P < 0.009) on days 2-10 postinoculation. The reduction in shedding of virus protein corresponded well with delayed onset of acute diarrhea (P < 0.02). Closer examination of tissue cross sections under electron microscopy revealed that the B. bifidum-ingested strain adhered to the epithelium of the small intestine. These results suggest that priming the intestine with B. bifidum is effective against experimental MRV challenge and confirmed the potential usefulness of this detector ELISA for studying the kinetics of group A rotavirus infection in animals and humans.

Animals↗
PubMed1995

Studies on the single-shelled rotavirus receptor with a synthetic peptide derived from the cytoplasmic domain of NS28.

The nonstructural glycoprotein NS28 of rotaviruses plays an important part in the assembly of double-shelled rotaviruses. C-terminal domains of the protein function as a receptor for single-shelled rotavirus particles at the membrane of the rough endoplasmic reticulum. In the present report we describe studies performed with a synthetic peptide corresponding to amino acid (aa) 160 to 169, the most hydrophilic C-terminal epitope of NS28. An antipeptide serum raised against this peptide demonstrated that this epitope was accessible in infected MA104 cells. Moreover, polymeric peptide was demonstrated to aggregate single-shelled rotavirus particles. This aggregation could be almost completely inhibited by preincubation with monomeric peptide. Our results clearly demonstrate that the epitope corresponding to aa 160-169 is able to bind single-shelled rotavirus particles.

Animals↗
PubMed1998

Oral delivery of homologous and heterologous strains of rotavirus to BALB/c mice induces the same profile of cytokine production by spleen cells.

In this work, we wanted to clarify if differences in antibody (Ab) and particularly in secretory immunoglobulin A (IgA) responses following homologous or heterologous rotavirus infection could be explained by different priming of specific T helper (Th) cells. We compared the Ab responses from suckling BALB/c mice orally inoculated with either a heterologous simian (SA11) or bovine (RF) rotavirus or a homologous murine rotavirus (EHPw), as well as the profile of cytokines produced by spleen cells after in vitro restimulation. Oral inoculation of EHPw and SA11 induced a similar pattern of Ab with mucosal and serum IgA associated with serum IgG with equal levels of IgG1 and IgG2a, whereas RF elicited a weak humoral response. We found that these strains induced the same mixed Th1/Th2 pattern of cytokine production by spleen cells with IFN-gamma and IL-5 as well as IL-10, but not IL-2 or IL-4. These findings suggest that the induction of immune response is probably not different between these strains. Other factors such as the amount of antigen, strain immunogenicity, and other cytokines, particularly produced in effector sites, remain to be considered in order to better explain the differences in secretory IgA following homologous or heterologous rotavirus infection.

Administration, Oral↗
PubMed1992

Characterization of full-length and polymerase chain reaction-derived partial-length Gottfried and OSU gene 4 probes for serotypic differentiation of porcine rotaviruses.

To determine the VP4 (P type) specificity of porcine rotaviruses, full- and partial-length gene 4 probes were produced from cloned Gottfried and OSU porcine rotavirus genomic segment 4 cDNAs. The gene 4 segments from the prototype Gottfried (VP7 serotype 4) and OSU (VP7 serotype 5) porcine rotavirus strains were selected for study because of their distinct P types and the occurrence of rotaviruses with similar serotypes among swine. Partial-length gene 4 cDNAs were produced and amplified by the polymerase chain reaction (PCR) and encompassed portions of the variable region (nucleotides 211 to 612) of VP8 encoded by genomic segment 4. The hybridization stringency conditions necessary for optimal probe specificity and sensitivity were determined by dot or Northern (RNA) blot hybridizations against a diverse group of human and animal rotaviruses of heterologous group A serotypes and against representative group B and C porcine rotaviruses. The PCR-derived gene 4 probes were more specific than the full-length gene 4 probes but demonstrated equivalent sensitivity. The Gottfried PCR-derived probe hybridized with Gottfried, SB2, SB3, and SB5 G serotype 4 porcine rotaviruses. The OSU PCR-derived probe hybridized with OSU, EE, A580, and SB-1A porcine rotaviruses and equine H1 rotavirus. Results of the hybridization reactions of the PCR-derived gene 4 probes with selected porcine rotavirus strains agreed with previous serological or genetic analyses, indicating their suitability as diagnostic reagents.

Animals↗
PubMed1986

Relationships between rotavirus diarrhea and intestinal microflora establishment in conventional and gnotobiotic mice.

Intestinal microflora did not play a role in the intensity or course of EDIM rotavirus-induced diarrhea, since similar results were observed in axenic and conventional mice. In conventional mice, rotavirus-induced diarrhea did not modify the establishment of Lactobacillus spp. and Escherichia coli before weaning. The consequences of diarrhea on the establishment of strictly anaerobic bacteria after weaning were studied through the measurement of two bacterial functions, the microbial barrier effect against E. coli and the development of the immunoglobulin A intestinal immune system. These two bacterial functions were expressed in a similar way in diarrheic and control mice. In young gnotobiotic mice inoculated with Clostridium perfringens or C. difficile, rotavirus infection led to an earlier development of both strains, as compared with controls. This effect was more pronounced with C. difficile. These results suggest that rotavirus infections might enhance opportunistic bacterial infections.

Animals↗
PubMed2005

[Changing patterns of communicable diseases in Korea].

Before twentieth centuries and during early twentieth centuries, communicable diseases were the major cause of morbidity and mortality in Korea. But reliable data are not available. After 1975, the overall morbidity and mortality from communicable diseases, rapidly declined. Recently many new pathogenic microbes were recognized: L. monocytogenes, Hantaan virus, Y. pseudotuberculosis, P. multocida, L. pneumophilia, Human immunodeficiency virus (HIV), G. seoi, H. capsulatum, C. burnetii, V. cholerae 0139, C. parvum, F. tularensis, E. coli 0157:H7, B. burgdorferi, S. Typhimurium DT104, Rotavirus, hepatitis C virus and so on. Since the first HIV infection recognized in 1985, the reported cases of infection and deaths from HIV/AIDS have been steady increased each year. Legionnaire's disease, E. coli 0157:H7 colitis, listeriosis and crytosporidiasis have been occurring just sporadically among immunocompromized cases. Many re-emerging communicable diseases were occurred in Korea: leptospirosis, malaria, endemic typhus, cholera, tsutsugamushi disease, salmonellosis, hepatitis A, shigellosis, mumps, measles, acute hemorrhagic conjunctivitis, brucellosis and so on. Leptospirosis and tsutsugamushi diseases have been noticed as major public health problems since 1980s. The malaria that had been virtually disappeared for a decade has reappeared from 1993 with striking increase of patients in recent 3-4 years. The distributions of salmonella and shigella serotypes have been changed a lot in recent few decades. Furthermore rapid emergence of antibiotic-resistant bacterial strains induces more difficult and complex problems in control of communicable diseases. We must recognize on the importance of environment and ecosystem conservation and careful prescription of anti-microbial agent in order to prevent communicable diseases.

Communicable Diseases, Emerging↗