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PubMed1982

Structural analysis of electrophoretic variation in the genome profiles of rotavirus field isolates.

Detailed structural studies were undertaken on five isolates of bovine rotavirus which showed variability in the migration patterns of their genome segments on electrophoresis in polyacrylamide gels. The individual genome segments of each isolate were characterized by partial digestion of terminally radiolabeled RNA with a base-specific nuclease. This analysis showed that whereas mobility variations were always associated with detectable changes in nucleotide sequence, sequence changes at least as great as those found in segments showing electrophoretic mobility variations were also detected in segments showing no mobility variation. Evidence for the occurrence of genome segment reassortment between viruses in the field was obtained from analyzing the species 11 RNAs from these five isolates. The overall conclusion from these results is that great care is required in the interpretation of simple genome profile analysis of different isolates for epidemiological purposes and that classification of these viruses solely on the basis of genome electropherotype could be misleading.

Animals↗
PubMed1982

Immunogenicity of type-specific C-mycoside glycopeptidolipids of mycobacteria.

Specific rabbit antibodies were obtained from the inoculation of a complex composed of methylated bovine serum albumin and the superficial type-specific C-mycoside glycopeptidolipid (GPL) antigens from serovar 20 of the Mycobacterium avium-M. intracellulare-M. scrofulaceum complex. Although the GPLs on their own are serologically active, they require the protein carrier for immunogenicity. Antibodies to the complex were detected by seroagglutination, gel diffusion, and indirect immunofluorescence. Cross-reaction was observed in the case of just one of 16 M. avium-M. intracellulare-M. scrofulaceum complex serovars tested (serovar 41). Double gel diffusion and immunoelectrophoresis showed two major antigen-antibody complexes in the reaction between anti-GPL-methylated bovine serum albumin and the GPL antigens. The studies of migration pattern and absorption indicated that immunoglobulin M was responsible for one of these complexes, whereas the antibody responsible for the second complex was unidentified. The specific anti-GPL serum is a useful tool for the identification and classification of atypical mycobacteria and for the study of the fate of the organisms and their superficies during phagocytosis.

Agglutination Tests↗
PubMed1983

Effective antibacterial protection induced by a Listeria monocytogenes-specific T cell clone and its lymphokines.

The capacity of the murine Listeria monocytogenes-specific T cell clone 9-36-1 and of lymphokines derived therefrom to induce antibacterial protection in vivo was studied. Clone 9-36-1 was stimulated to proliferate and to produce lymphokines by in vitro culture with syngeneic accessory cells and heat-killed L. monocytogenes. Although 9-36-1 cells were highly active in vitro, intravenous transfer of the cells resulted in marginal protection against a systemic infection with L. monocytogenes. In contrast, 9-36-1 cells injected subcutaneously together with L. monocytogenes into the footpad induced marked protection in syngeneic, but not in allogeneic, mice. Multiplication of Salmonella typhimurium was not reduced by the T cell clone. Studies with 51Cr-labeled T cells indicated that the low activity of intravenously transferred cells was due to an altered migration pattern. Lymphokines produced by 9-36-1 cells in vitro induced protection against L. monocytogenes in syngeneic recipient mice. Lymphokine-induced protection was also demonstrable in allogeneic recipients and against S. typhimurium. These findings suggest that the L. monocytogenes-specific T cell clone 9-36-1, although unable to immigrate into sites of bacterial deposition, had retained its ability to mobilize antibacterial defense mechanisms once present at the site of reaction.

Animals↗
PubMed1983

Isolation, propagation, and characterization of a second equine rotavirus serotype.

A rotavirus designated strain H-2 was isolated in primary African green monkey kidney cells from a foal with diarrhea. This cell culture-adapted strain was found to be similar, if not identical, to simian rotavirus (strains MMU18006 and SA-11) and canine rotavirus (strain CU-1) and, in addition, demonstrated a one-way antigenic relationship with five human rotavirus strains (P, B, no. 14, no. 15, and YO) of the third human rotavirus serotype by the plaque reduction neutralization test. This is the fifth example of an animal rotavirus which shares serotypic specificity with a human rotavirus. The H-2 strain is distinct from the H-1 strain (Y. Hoshino et al., J. Clin. Microbiol., in press) of equine rotavirus not only in serotypic specificity by neutralization but also in subgroup specificity, hemagglutinating activity, and RNA electrophoretic migration pattern, thus establishing the existence of a second equine rotavirus serotype. This H-2 isolate is also distinct by neutralization from three other human rotavirus serotypes, 1 (Wa), 2 (DS-1), and 4 (St. Thomas no. 4), as well as bovine (NCDV), and porcine (OSU) rotaviruses.

Animals↗
PubMed1987

Allergenic and blastogenic reactivity of three antigens from Mycobacterium tuberculosis in sensitized guinea pigs.

Three antigens from a culture filtrate of Mycobacterium tuberculosis H37Rv were purified by affinity chromatography, using monoclonal antibodies. The molecular weights of the purified antigens are 17,000 to 19,000, 32,000 to 33,000, and 39,000, respectively, and by their migration patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, they all appeared to be single-chain polypeptides. Western blot and enzyme-linked immunosorbent assay analyses indicated that the antigens are non-cross-reactive. All antigens generated an intermediate to strong skin reaction when tested in guinea pigs previously immunized with a live M. bovis BCG vaccine or with an oil emulsion preparation of phenol-or heat-killed M. tuberculosis. Lymphocytes isolated from peripheral blood or lymph nodes of similarly immunized guinea pigs could be stimulated by purified protein derivative and the purified antigens. Qualitative differences in stimulatory capacity between the preparations were demonstrated. The antigens may prove useful in further studies of the immunology and pathogenesis of tuberculosis.

Animals↗
PubMed1987

Phenotypic variation in epitope expression of the Neisseria gonorrhoeae lipooligosaccharide.

Gonococcal lipooligosaccharides (LOSs) are a series of antigenically complex heteropolymers. To investigate whether all members of clonally selected populations of Neisseria gonorrhoeae express antigenically similar LOS, we studied gonococcal strains 4505 and 220 with monoclonal antibodies 6B4 and 3F11 which have specificity for different oligosaccharide epitopes on the same or comigrating LOS unit(s) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Fluorescent-antibody and immunoelectron microscopy studies indicated that all members of the clonally selected populations were not homogenous for the epitopes these antibodies recognized. Fluorescence-activated cell sorting studies of 3F11-coated strain 220 indicated that the density of epitope expression was a function of time of growth. The population could be separated into two broad groups corresponding to organisms staining strongly or weakly for the 3F11 epitope, and the epitope density decreased during the late-log and stationary phases of growth. Sequentially staining organisms on Formvar grids with 6B4 and 3F11, followed by staining with either 5- or 15-nm colloidal gold spheres conjugated to goat anti-mouse immunoglobulin M demonstrated the following populations of cells among organisms derived from a single clone: organisms which stained for both 6B4 and 3F11 epitopes and organisms which stained for either 6B4 epitopes alone or 3F11 epitopes alone. Immunofluorescence microscopy studies with rhodamine and fluorescein goat anti-mouse immunoglobulin M conjugates sequentially staining organisms on Formvar grids with 3F11 and 6B4 also demonstrated these three populations. Analysis of LOS preparations made over the last 5 years indicated no change in serotype antigen concentration or in sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration pattern. These studies indicate that while clonally selected strains of Neisseria gonorrhoeae undergo phenotypic variation at the epitope level, the impact of this variation on the total LOS of the population has little overall effect on its antigenic or physicochemical properties.

Antibodies, Monoclonal↗
PubMed1990

Genomic variation of Trypanosoma cruzi: involvement of multicopy genes.

By using improved pulsed field gel conditions, the karyotypes of several strains of the protozoan parasite Trypanosoma cruzi were analyzed and compared with those of Leishmania major and two other members of the genus Trypanosoma. There was no difference in chromosome migration patterns between different life cycle stages of the T. cruzi strains analyzed. However, the sizes and numbers of chromosomal bands varied considerably among T. cruzi strains. This karyotype variation among T. cruzi strains was analyzed further at the chromosomal level by using multicopy genes as probes in Southern hybridizations. The chromosomal location of the genes encoding alpha- and beta-tubulin, ubiquitin, rRNA, spliced leader RNA, and an 85-kilodalton protein remained stable during developmental conversion of the parasite. The sizes and numbers of chromosomes containing these sequences varied among the different strains analyzed, implying multiple rearrangements of these genes during evolution of the parasites. During continuous in vitro cultivation of T. cruzi Y, the chromosomal location of the spliced leader gene shifted spontaneously. The spliced leader gene encodes a 35-nucleotide RNA that is spliced in trans from a 105-nucleotide donor RNA onto all mRNAs in T. cruzi. The spliced leader sequences changed in their physical location in both the cloned and uncloned Y strains. Associated with the complex changes was an increase in the infectivity of the rearranged variant for tissue culture cells. Our results indicate that the spliced leader gene clusters in T. cruzi undergo high-frequency genomic rearrangements.

Animals↗
PubMed1997

High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets.

Mycobacterium tuberculosis, the primary etiologic agent of tuberculosis, is the world's leading cause of death from a single infectious agent, and new vaccines and drugs to combat it are urgently needed. The major extracellular proteins of M. tuberculosis, which are released into its phagosome in macrophages, its host cells in humans, are leading candidates for a vaccine and prime targets for new drugs. However, the development of these biologicals has been hampered by the unavailability of large quantities of recombinant extracellular proteins identical to their native counterparts. In this report, we describe the heterologous expression and secretion of four major M. tuberculosis extracellular proteins (the 30-, 32, 16-, and 23.5-kDa proteins--the first, second, third, and eighth most abundant, respectively) in rapidly growing, nonpathogenic mycobacterial species. Multiple attempts to obtain secretion of the proteins by using Escherichia coli- and Bacillus subtilis-based expression systems were unsuccessful, suggesting that high-level expression and secretion of these Mycobacterium-specific proteins require a mycobacterial host. All four recombinant proteins were stably expressed from the cloned genes' own promoters at yields that were 5- to 10-fold higher than those observed for the native proteins. The four proteins were purified to apparent homogeneity from culture filtrates by ammonium sulfate precipitation and ion-exchange and molecular sieve chromatography. The recombinant proteins were indistinguishable from their native counterparts by multiple criteria. First, N-terminal amino acid sequence determination demonstrated that processing of the leader peptides was highly accurate. Second, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed identical migration patterns. Third, mass spectrometry analysis confirmed that differences in mass were < or = 5 Da. A homolog of the M. tuberculosis 30-kDa protein was identified in M. smegmatis by means of DNA analyses and immunoscreening. This is the first time that secretion of recombinant M. tuberculosis extracellular proteins in their native form has been achieved. This study opens the door to mass production of correctly processed and secreted extracellular proteins of M. tuberculosis in a heterologous host and allows ready evaluation of their biologic and immunologic function.

Amino Acid Sequence↗
PubMed1997

Structurally defined epitopes of Haemophilus ducreyi lipooligosaccharides recognized by monoclonal antibodies.

By use of enzyme-linked immunosorbent assay and immunoblotting techniques, the migration patterns and binding epitopes of lipooligosaccharides (LOS) from 10 Haemophilus ducreyi strains were investigated with two monoclonal antibodies (MAbs), MAHD6 and MAHD7, raised against LOS from H. ducreyi ITM 2665. Closely related LOS, with defined structures, from Haemophilus influenzae, Bordetella pertussis, Aeromonas spp., and synthetic glycoproteins were also included in the analyses. The MAbs bound to conserved epitopes of LOS exposed on the surface of H. ducreyi. The MAb MAHD6 reacted with 8 of the 10 LOS from H. ducreyi but with none of the other Haemophilus or Bordetella spp. with structurally defined LOS. It is suggested that MAb MAHD6 binds to a LOS epitope (-DD-Hepp-1-->6-beta-D-Glcp-). This LOS epitope is not present in the hexasaccharide structure of LOS from H. ducreyi ITM 4747 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J. A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Because MAb MAHD6 reacts with the epitope mentioned above, it also discriminates between the two LOS structures, the hexasaccharide group and the nonasaccharide group, of H. ducreyi strains. MAb MAHD7 recognizes the common conserved inner core region of the LOS because it reacts with all H. ducreyi strains and with LOS with minor components in the inner core epitope structure. Rabbit polyclonal sera raised against the LOS from strains CCUG 4438 and CCUG 7470 were tested with the 10 LOS from the H. ducreyi strains. The antiserum to CCUG 7470 reacted with all H. ducreyi strains as did MAb MAHD7, whereas the antiserum to CCUG 4438 reacted with only its homologous strain and strain ITM 4747. Also, the LOSs of our reference strains CCUG 4438 and CCUG 7470 were structurally analyzed by use of sugar analyses and electrospray ionization-mass spectrometry. The hexasaccharide and nonasaccharide structures obtained from LOS of strains CCUG 4438 and CCUG 7470 were identical to the described LOS structures from H. ducreyi ITM 4747 and ITM 2665, respectively. In conclusion, the MAb MAHD6 recognizes an epitope present in the nonasaccharide LOS group, whereas the MAb MAHD7 recognizes a conserved epitope on LOS of H. ducreyi, which is present in all strains of H. ducreyi tested. Two major groups of oligosaccharides were distinguished by their LOS structures and the reactivity of monoclonal as well as polyclonal antibodies. The majority of H. ducreyi strains possess a nonasaccharide structure of LOS.

Animals↗
PubMed2001

Soluble pilin of Neisseria gonorrhoeae interacts with human target cells and tissue.

Pili of Neisseria gonorrhoeae are phase-variable surface structures that mediate adherence to host target cells. Each pilus is composed of thousands of major pilus subunits, pilins, pilus-associated protein PilC, and possibly other components. Piliated and nonpiliated gonococcal clones may secrete a soluble smaller pilin (S-pilin) that is cleaved after amino acid 39 of the mature pilin protein. Here, purified S-pilin was found to migrate as a 61- to 64-kDa double band on nondenaturing gels, suggesting the formation of tetrameric S-pilin proteins with two isomeric forms. In situ studies of binding to formalin-fixed tissue sections demonstrated the binding of S-pilin to human tissue but not to tissue from mouse or rat organs, showing the presence of a human-specific receptor-binding domain within the pilin polypeptide. Pretreatment of the target tissues with proteinase K decreased gonococcal binding dramatically, whereas pretreatment with neuraminidase and meta-periodate, which cleave carbon-carbon linkages between vicinal hydroxyl groups in carbohydrates, did not affect gonococcal binding. In overlay assays, purified S-pilin bound to a band with a migration pattern and size similar to those of CD46, a cellular pilus receptor. Further, binding of N. gonorrhoeae to target cells and tissues could be blocked by both CD46 antibodies and purified S-pilin. These data argue that S-pilin interacts with a protein domain(s) of the CD46 receptor on human cells.

Acrylic Resins↗
PubMed2003

Differential production of macrophage inflammatory protein 1gamma (MIP-1gamma), lymphotactin, and MIP-2 by CD4(+) Th subsets polarized in vitro and in vivo.

Due to differential expression of chemokine receptors, the Th1 and Th2 subsets of CD4(+) T cells differ in their migratory responses to chemokines. These differences in the migration patterns are likely to play a role in the initiation and regulation of Th1 and Th2 immune responses, inflammatory processes, and T-cell-mediated pathology. In the present study we evaluated the role of activated Th cells as producers of chemokines. Three different sources of murine Th cells were used, i.e., long-term-cultured Th1 and Th2 cell clones, Th1 and Th2 cells differentiated from naïve CD4(+) spleen and lymph node cells in vitro, and Th1 and Th2 subsets polarized in vivo using a murine experimental Leishmania major infection model. Following stimulation with anti-CD3, macrophage inflammatory protein 1gamma (MIP-1gamma) and lymphotactin were produced selectively by Th1 cells but not by Th2 cells. In contrast, only Th2 cells produced MIP-2. The possible biological relevance of these data was substantiated by the finding that in vivo-polarized Th1 cells, but not Th2 cells, produced MIP-1gamma and lymphotactin while in vivo-polarized Th2 cells secreted MIP-2. The above data demonstrate that Th1 and Th2 cells differ in their ability to produce chemokines, suggesting that Th1 and Th2 subsets differentially contribute to recruitment of cells into inflammatory foci.

Animals↗
PubMed1972

Molecular heterogeneity of the Bdellovibrios: evidence of two new species.

A systematic examination of a variety of isolates of the bacterial endoparasite Bdellovibrio has revealed extensive molecular diversity. The quantity of deoxyribonucleic acid (DNA) polynucleotide homology ranges from more than 90% among the isolates with DNA containing 50 to 51% guanine plus cytosine (GC) to undetectable levels between the 43% GC and 51% GC isolates. The two isolates with low GC-containing DNA (H-I Bdellovibrio A3.12 and UKi2) have only 16% DNA homology. H-I Bdellovibrio A3.12 and 109 have barely detectable ribosomal ribonucleic acid (rRNA) homology, whereas the homology approaches 100% among all the high GC isolates tested. Cases of high DNA/DNA and DNA/rRNA homologies are reflected in low dissimilarities of enzyme migration patterns in starch gel electrophoresis. The dissimilarities exhibited among the high GC Bdellovibrio isolates are as low as those previously reported for different Escherichia coli strains. The zymograms of H-I Bdellovibrio A3.12 and UKi2 are completely different from each other as well as from all other bdellovibrios (100% dissimilarity). Genome sizes determined for the representative isolates demonstrate three size ranges which coincide with group differences based on the above measurements. Enzyme assays reveal that all isolates possess a tricarboxylic acid cycle and most contain an alanine and glutamic dehydrogenase. We conclude that the use of bacterial endoparasitism as a defining trait has resulted in a molecularly diverse collection of isolates. It is recommended that the specific epitaph bacteriovorus be used only for the type specimen (Bdellovibrio 100 of Stolp and Starr, 1963) and for other related 50 to 51% GC isolates. The heterogeneity of the group warrants two new species. We designate Bdellovibrio A3.12 as the nomenclatural type of B. starrii sp. n. and Bdellovibrio UKi2 as the nomenclatural type of B. stolpii sp. n.

Adenine Nucleotides↗
PubMed2000

An abundant DNA binding protein from the hyperthermophilic archaeon Sulfolobus shibatae affects DNA supercoiling in a temperature-dependent fashion.

The DNA binding protein Ssh10b, a member of the Sac10b family, has been purified from the hyperthermophilic archaeon Sulfolobus shibatae. Ssh10b constitutes about 4% of the cellular protein. Electrophoretic mobility shift assays showed that Ssh10b first bound a double-stranded DNA fragment with an estimated binding size of approximately approximately 12 bp, forming distinct shifts, until the DNA was coated with the protein. Binding of more Ssh10b resulted in the formation of smears of lower mobilities. The migration pattern of the smearing Ssh10b-DNA complexes was affected by temperature, whereas that of complexes associated with the distinct shifts was not. Interestingly, Ssh10b was capable of constraining negative DNA supercoils in a temperature-dependent fashion. While the ability of the protein to constrain supercoils was weak at 25 degrees C, it was enhanced substantially at 45 degrees C or higher temperatures (up to 80 degrees C). Taken together, our data suggest that archaeal proteins of the Sac10b family may affect the topology of chromosomal DNA in thermophilic archaea at their growth temperatures.

Amino Acid Sequence↗
PubMed1982

Isolation of human rotaviruses in primary cultures of monkey kidney cells.

We succeeded in isolating human rotaviruses from the feces of gastroenteritis patients by using roller cultures of primary cynomolgus monkey kidney cells with trypsin in the maintenance medium but without concentration and trypsin treatment of the inocula at each passage level. These cells were found to be more sensitive than MA-104 cells (derived from fetal rhesus monkey kidney) for the propagation of human rotaviruses. Polyacrylamide gel electrophoresis of the genome RNA revealed that there were small differences in the migration pattern of the segments among all the strains isolated from 1976 to 1981. The cultivation of human rotaviruses in primary cell cultures might aid in developing a liver rotavirus vaccine.

Animals↗
PubMed1983

Isolation and characterization of an equine rotavirus.

A rotavirus, designated as the H-1 strain, was isolated from a diarrheic foal in primary African green monkey kidney cells and MA104 cells. This cell culture-adapted strain hemagglutinated erythrocytes of human group O, rhesus monkeys, guinea pigs, and sheep. It was found to be similar, if not identical, to porcine rotaviruses (strains OSU, EE, and A-580) by plaque reduction neutralization and hemagglutination inhibition tests, and, in addition, it was found to belong to subgroup 1. This equine rotavirus has an RNA electrophoretic migration pattern which was distinct from those of the three strains of porcine rotavirus. The serological relationship established by plaque reduction neutralization and hemagglutination inhibition tests between the equine (H-1) and porcine (OSU, EE, and A-580) rotaviruses is an example of a rotavirus of the same serotype being isolated from different species. The H-1 strain was distinct from four human rotavirus serotypes (Wa, DS-1, P, and St. Thomas 4) as well as from bovine rotavirus NCDV, simian rotavirus MMU18006, and canine rotavirus CU-1 by plaque reduction neutralization tests. This equine isolate (H-1) was found to be related antigenically to canine CU-1 and bovine NCDV rotaviruses in a one-way fashion by hemagglutination inhibition tests.

Animals↗
PubMed1986

Some antigenic properties of Haemophilus parasuis and a proposal for serological classification.

We propose a serological classification of Haemophilus parasuis into at least five serovars, using an agar-gel-precipitation test with extracts from autoclaved cells. Thirty-two strains were examined, and it was possible to classify 26 of them. The specific antigens were thermostable and soluble and were not affected by pronase treatment but could be extracted by phenol, suggesting a polysaccharide. This polysaccharide seemed to be identical with the capsular substance of serovars 1, 2, and 3. In the presumably uncapsulated strains of serovars 4 and 5, the specific antigen was probably located in the outer membrane. The diversity of the specific substance within the different serotypes was shown by the differences in their electrophoretic migration patterns. Other extraction procedures showed that the washing supernatant and extracts at 60 and 100 degrees C were identical with the 121 degrees C extracts for serovars 1, 2, and 3. In serovars 4 and 5, washing antigens, if present, were different from 100 and 121 degrees C extracts. Other common antigens, presumably proteinaceous antigens, were detected after extraction at 60 and 100 degrees C. The slide and tube agglutination tests allowed classification only for the capsulated strains of serovars 1, 2, and 3. The specific agglutinogens were very sensitive to incubation temperature, and the absorption test showed them to be identical with the 121 degrees C precipitinogens.

Agglutination Tests↗
PubMed1988

Serial propagation of porcine group C rotavirus (pararotavirus) in a continuous cell line and characterization of the passaged virus.

The Cowden strain of porcine group C rotavirus (pararotavirus) was adapted to serial passage in a continuous monkey kidney cell line (MA104). Key factors in its successful adaptation included use of virus passaged in primary porcine kidney cells as the initial inoculum, use of roller tubes, and addition of pancreatin to the maintenance medium. A cell culture immunofluorescence test was used to quantitate the virus at each passage level, since a possible cytopathic effect was obscured by the effects of pancreatin. The virus titers dropped after initial passage into MA104 cells but increased thereafter, with peak titers evident after 16 passages (10(7) immunofluorescence U/ml). Immune electron microscopy and genome electropherotyping were used to identify group C rotavirus particles and confirm group C rotavirus double-stranded RNA gel migration patterns, respectively, from infected cell culture supernatants. The electropherotype of the cell culture-propagated group C rotavirus was identical to that of the gut virulent virus from which it was derived. The cell culture-passaged group C rotavirus also retained its infectivity for gnotobiotic pigs. No group A rotavirus was detected in the intestinal contents of the pigs or in cell culture fluids from group C rotavirus-inoculated monolayers with the two former techniques or the cell culture immunofluorescence test. This is the first verified report of serial propagation of a non-group A rotavirus in a continuous cell line.

Animals↗
PubMed1989

Comparative studies of the antigenic polypeptide species VP4, VP6, and VP7 of three strains of bovine rotavirus.

Three bovine rotavirus strains belonging to two distinct serotype groups, serotype 6 (NCDV and B641) and B223, distinct from the other six mammalian rotavirus serotypes but not yet assigned to a serotype group, were compared with each other and with canine rotavirus (K9, serotype 3) by studying the properties of their cognate polypeptide species VP4, VP6, and VP7. The three viruses showed distinct differences in the polyacrylamide gel electrophoretic migration rates of protein species VP4 and VP7, with minor differences in VP6. Differences were also observed among the migration patterns of genome segments 4, 6, and the 7-8-9 triplet, which encode VP4, VP6, and VP7, respectively. Monoclonal antibodies (MAbs) to B223, which were directed against VP4 or VP7, showed homologous specificity for neutralization and immunofluorescence (IF), although one MAb reactive with VP4 also reacted by IF and by immunoprecipitation (IP) with all four viruses and weakly neutralized B641 and K9. This MAb may react with the epitope responsible for the B223-induced one-way neutralizing and protection response of calves against B641 observed in earlier studies. MAbs reactive with VP6 by IP showed enzyme-linked immunosorbent assay and IF reactivity with all three bovine viruses and the canine virus. The two serotype 6 viruses could be distinguished by the two B641 MAbs, B641-N2b reacting by neutralization and IF with both viruses and B641-N1 reacting with B641 and the serotype 3 canine rotavirus but not with NCDV. One nonneutralizing B641 MAb reacted by IP and IF with VP7 of all four rotaviruses examined, and one B223 MAb neutralized B223 and, to low titer, B641 and K9 although reacting by IP and IF with all four viruses. Three MAb-resistant mutants were selected by passage of B223 in the presence of one of three selected B223 MAbs at concentrations which only neutralized approximately 90% of the infectious virions. The resulting mutants were 100% resistant to neutralization with their respective MAb but remained neutralizable by the same selection of MAbs as the parent B223 virus.

Animals↗