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S Urasawa

Publications and source records attributed to S Urasawa.

At least 109 records · Page 6Linked to original sources
PubMed1988

Prediction of human rotavirus serotype by nucleotide sequence analysis of the VP7 protein gene.

Human rotavirus field isolates were characterized by direct sequence analysis of the gene encoding the serotype-specific major neutralization protein (VP7). Single-stranded RNA transcripts were prepared from virus particles obtained directly from stool specimens or after two or three passages in MA-104 cells. Two regions of the gene (nucleotides 307 through 351 and 670 through 711) which had previously been shown to contain regions of sequence divergence among rotavirus serotypes were sequenced by the dideoxynucleotide method with two different synthetic oligonucleotide primers. The resulting nucleotide sequences were compared with the corresponding sequences from rotaviruses of known serotype (serotype 1, 2, 3, or 4). A total of 25 field isolates and 10 laboratory strains examined by this method exhibited marked sequence identity in both areas of the gene with the corresponding regions of 1 of the 4 reference strains. In addition, the predicted serotype from the sequence analysis correlated in each case with the serotype determined when the rotaviruses were examined by plaque reduction neutralization or reactivity with serotype-specific monoclonal antibodies. These data suggest that as a result of the high degree of sequence conservation observed among rotaviruses of the same serotype, it is possible to predict the serotype of a rotavirus isolate by direct sequence analysis of its VP7 gene.

Amino Acid Sequence↗
PubMed1988

Cross-reactive and serotype-specific neutralization epitopes on VP7 of human rotavirus: nucleotide sequence analysis of antigenic mutants selected with monoclonal antibodies.

The neutralization epitopes of human and simian rotavirus protein VP7 were studied by producing six neutralizing monoclonal antibodies (N-MAbs) and using these N-MAbs to select antigenic mutants that resisted neutralization by the N-MAbs used for their selection. Cross-neutralization tests between the N-MAbs and the antibody-selected antigenic mutants identified one cross-reactive and five distinct serotype-specific neutralization epitopes which operationally overlapped one another and constituted a single antigenic site. In addition, the amino acid substitutions in human rotavirus VP7 that are responsible for the antigenic alterations in the mutants selected with anti-VP7 cross-reactive or serotype-specific N-MAbs were identified. All the amino acid substitutions in the antigenic mutants occurred in one of two variable regions: amino acids 87 to 101 and 208 to 221.

Amino Acid Sequence↗
PubMed1988

Identification of cross-reactive and serotype 2-specific neutralization epitopes on VP3 of human rotavirus.

The group A rotaviruses are composed of at least seven serotypes. Serotype specificity is defined mainly by an outer capsid protein, VP7. In contrast, the other surface protein, VP3 (775 amino acids), appears to be associated with both serotype-specific and heterotypic immunity. To identify the cross-reactive and serotype-specific neutralization epitopes on VP3 of human rotavirus, we sequenced the VP3 gene of antigenic mutants resistant to each of seven anti-VP3 neutralizing monoclonal antibodies (N-MAbs) which exhibited heterotypic or serotype 2-specific reactivity, and we defined three distinct neutralization epitopes on VP3. The mutants sustained single amino acid substitutions at position 305, 392, 433, or 439. Amino acid position 305 was critical to epitope I, whereas amino acid position 433 was critical to epitope III. In contrast, epitope II appeared to be more dependent upon conformation and protein folding because both amino acid positions 392 and 439 appeared to be critical. These four positions clustered in a relatively limited area of VP5, the larger of the two cleavage products of VP3. At the positions where amino acid substitutions occurred, there was a correlation between amino acid sequence homology among different serotypes and the reactivity patterns of various viruses with the N-MAbs used for selection of mutants. A synthetic peptide (amino acids 296 to 313) which included the sequence of epitope I reacted with its corresponding N-MAb, suggesting that the region contains a sequential antigenic determinant. These data may prove useful in current efforts to develop vaccines against human rotavirus infection.

Amino Acid Sequence↗
PubMed1987

Different virus-precipitating activities of neutralizing monoclonal antibodies that recognize distinct sites of poliovirus particles.

Two neutralizing monoclonal antibodies (4 C 4 and 4 F 2) against type 1 poliovirus, Mahoney strain, recognized distinct antigenic sites of the virus particles; 4 C 4 antibody bound to vertices of native and heated (56 degrees C, 30 minutes) virus of Mahoney strain, while 4 F 2 antibody reacted with specific surface protrusions of native virus of Mahoney and Sabin strains. The difference in the location of neutralization epitopes with which the two antibodies react was confirmed in the neutralization reaction by the use of mutants resistant to 4 C 4 and 4 F 2 antibody. In immune electron microscopy, double immunodiffusion and sucrose density gradient analysis of virus-antibody complexes, the two antibodies showed a marked difference in their virus-precipitating activities. The 4 C 4 antibody recognizing vertices of the virus particle had little virus-precipitating activity. In contrast, the 4 F 2 antibody that bound to specific surface protrusions of native virus aggregated virus particle efficiently. In neutralization assays, however, the 4 C 4 antibody exhibited a slightly stronger neutralizing activity than the 4 F 2 antibody. Thus, it was suggested that the strength in precipitating activities of the two antibodies did not correlate with that in their neutralizing activities.

Antibodies, Monoclonal↗
PubMed1987

Direct serotyping of human rotavirus in stools by an enzyme-linked immunosorbent assay using serotype 1-, 2-, 3-, and 4-specific monoclonal antibodies to VP7.

Four serotypes of human rotaviruses that can be differentiated by neutralization tests have been described so far. We prepared serotype 1-, 2-, 3-, and 4-specific, neutralizing monoclonal antibodies to human rotaviruses. All were directed to VP7, a glycoprotein that carries a major serotype specificity. An enzyme-linked immunosorbent assay using these monoclonal antibodies has been developed for serotyping human rotavirus isolates. The sensitivity and specificity of this method were verified by using various strains of human rotavirus that were adapted to cell culture. Furthermore, it was shown that the method was applicable to serotyping human rotaviruses directly in stool specimens.

Antibodies, Monoclonal↗
PubMed1987

Antigenic characterization of rotaviruses isolated in Kenya from 1982 to 1983.

The electropherotypes of human rotavirus RNAs from 100 diarrheic stool specimens collected in two major districts of Kenya from 1982 to 1983 were previously reported (Y. Chiba, C. Miyazaki, Y. Makino, L. N. Mutanda, A. Kibue, E. O. Lichenga, and P. M. Tukei, J. Clin. Microbiol. 19:579-582, 1984). Of these specimens, 25 that contained rotaviruses with different RNA electropherotypes were subjected to a virus isolation experiment with MA-104 cells, and 16 rotavirus strains were isolated. The use of an enzyme-linked immunosorbent assay with subgroup-specific monoclonal antibodies enabled us to successfully subgroup 15 isolates: 4 in subgroup I and 11 in subgroup II. By fluorescent-focus-neutralization test with serotype-specific rabbit antisera, 13 isolates could be serotyped: 7 as serotype 1, 4 as serotype 2, and 2 as serotype 3. Of the remaining three isolates, F153, F247, and G402, the former was doubly neutralizable with serotype 1 and serotype 4 antisera and the latter two were neutralizable with serotype 3 and serotype 4 antisera. Detailed analysis with the antisera against F153 and F247 and four serotype-specific, VP7-directed monoclonal antibodies suggested that F153 is a serotypic mosaic strain with serotype 4-specific VP3 and serotype 1-specific VP7 outer capsid proteins and F247 and G402 are possibly antigenic mosaic strains with serotype 3 and serotype 4 antigens. On the basis of the correspondence of the rotavirus isolate serotypes determined in this study to the electropherotypes reported previously, it was inferred that serotype 1 strains were most prevalent in two districts of Kenya from 1982 to 1983, followed by any type of serotypic mosaic strains.

Antibodies, Monoclonal↗
PubMed1987

Genetic relatedness among human rotavirus genes coding for VP7, a major neutralization protein, and its application to serotype identification.

Antigenic characterization of human rotaviruses by plaque reduction neutralization assay has revealed four distinct serotypes. The outer capsid protein VP7, coded for by gene 8 or 9, is a major neutralization protein; however, studies of rotaviruses derived from genetic reassortment between two strains have confirmed that another outer capsid protein, VP3, is in some cases equally important in neutralization. In this study, the genetic relatedness of the genes coding for VP7 of human rotaviruses belonging to serotypes 1 through 4 was examined by hybridization of their denatured double-stranded genomic RNAs to labeled single-stranded mRNA probes derived from human-animal rotavirus reassortants containing only the VP7 gene of their human rotavirus parent. A high degree of homology was demonstrated between the VP7 genes of strain D and other serotype 1 human rotaviruses, strain DS-1 and other serotype 2 human rotaviruses, strain P and other serotype 3 human rotaviruses, and strain ST3 and other serotype 4 human rotaviruses. Hybrid bands could not be demonstrated between the VP7 gene of D, DS-1, P, or ST3 and the corresponding gene of human rotaviruses belonging to a different serotype. RNA specimens extracted from the stools of 15 Venezuelan children hospitalized with rotavirus diarrhea were hybridized to each of the reassortant probes representing the four human serotypes. All five viruses with short RNA patterns showed homology with the DS-1 strain VP7 gene; two of these were previously adapted to tissue culture and shown to be serotype 2 strains by tissue culture neutralization. Of the remaining 10 viruses with long RNA patterns, 2 hybridized only to the D strain VP7 gene, 6 hybridized only to the P strain VP7 gene, and 2 hybridized only to the ST3 strain VP7 gene. Hybridization using single human rotavirus gene substitution reassortants as probes may provide an alternative method for identifying the VP7 serotype of field isolates that would circumvent the need for tissue culture adaptation.

Capsid↗
PubMed1987

Cross-reactive neutralization epitopes on VP3 of human rotavirus: analysis with monoclonal antibodies and antigenic variants.

We analyzed cross-reactive neutralization epitopes on protein VP3 of human rotavirus (HRV) by the use of neutralizing monoclonal antibodies (N-MAbs), which showed a variety of interserotypic reactivity patterns when examined in a neutralization test and an enzyme-linked immunosorbent assay against 15 HRV and 2 animal RV strains. Serological study with the six cross-reactive N-MAbs revealed antigenic variations in some HRV strains within the same serotype as well as a marked antigenic difference between serotype 2 strains and serotype 1, 3, and 4 strains. Epitope analysis of the antigenic variants resistant to the six individual cross-reactive N-MAbs suggested the existence of at least three distinct cross-reactive neutralization epitopes on VP3 of HRV.

Antibodies, Monoclonal↗
PubMed1987

Neoplastic transformation of Chinese hamster cells in vitro after treatment with flavoring agents.

Chinese hamster B241 cells were treated with 5 nM allylisothiocyanate (AI) or 10 nM trans-cinnamaldehyde (CA) and surviving cells were cultivated for generations until the cells acquired the characteristics of transformed cells based on in vitro criteria: increase in (a) saturation density in monolayer culture, (b) plating efficiency at low serum level and (c) colony forming efficiency (CFE) in soft agar medium. When the values of CFE of the treated cells had become significantly high, anchorage-independent colonies were isolated, propagated and then subjected to an assay for neoplastic transformation. The anchorage-independent clonal cells (CH-AI-AG+-1 and CH-CA-AG+-2) were subcutaneously injected into a suprascapular site in nude mice. The mice were maintained in an SPF animal care facility and observed for tumor formation. Growth of neoplasm at the injection sites was observed in 6 out of 7 mice and in all of the 6 mice injected with CA- and AI-transformed cells, respectively, during 3 to 8 months after the injection, as compared with 1 out of 6 mice injected with untreated control cells. Subsequent transplantation of the tumor cells into new mice induced tumor production at the injection site in all the animals within a considerably shorter period of time than that following the initial inoculation. Malignancy of the neoplastic cells was ascertained by histological examination, and the cells were found to have karyotypes of the hamster cells after in vitro cultivation of the tumor cells. These experimental results suggest the transforming potency of the flavoring agents in Chinese hamster cells.

Acrolein↗
PubMed1987

Induction of mutagenic activity of tetracycline by synergistic action with nitrite.

Synergistic mutagenicity of tetracycline(TC) and nitrite was investigated by the bacterial mutation test in the Salmonella/microsome system by using the reaction products obtained under neutral condition as well as under acidic condition (in simulated gastric juice). Results from tests using Salmonella typhimurium TA 100 disclosed a significant increase in the appearance of histidine (+) (his+) revertants by the reaction product between TC and nitrite in the presence of the rat liver-microsomal enzyme system (S9 mix), while the mutagenic potency of the reaction product of the two compounds in simulated gastric juice was extremely weak. In the process of the reaction of TC with and without nitrite in the presence of S9 mix, formaldehyde was detected, indicating the demethylation of TC by demethylase in S9. To explain the induction mechanism of the synergistic mutagenicity of TC and nitrite, it was suggested that the alkylating reaction of nitroso compound formed by nitrosation of the 4-demethylated intermediate of TC by the aid of microsomal metabolism is more important than the well known nitrosation mechanism under acidic condition such as in gastric juice in rats and in simulated gastric juice.

Animals↗
PubMed1987

Comparison of naturally occurring poliovirus-reactive immunoglobulins in bovine and equine sera.

Bovine and equine sera were screened for poliovirus-reactive immunoglobulins (PRIgs) by means of neutralization and precipitation reactions with type 1 poliovirus. Bovine serum B1826 and B36 were found to contain such PRIgs from their reactivity to various PRIgs-resistant mutants of type 1 poliovirus origin. Neutralization and precipitation reactions with six mono-specific antibodies obtained by absorbing antiserum with each of the six different PRIgs-resistant virus mutants revealed that three antibodies were active in precipitation reaction while the others were substantially ineffective. On the basis of the results obtained and the findings reported to date, the mechanism of production of PRIgs in bovine and equine sera was discussed.

Animals↗
PubMed1986

Protective effect of naturally acquired homotypic and heterotypic rotavirus antibodies.

To assess serotype specificity of immune resistance to rotavirus gastroenteritis, the relation between pre-existing neutralising antibodies to homotypic and heterotypic rotaviruses and protection against infection or clinical illness was investigated. The subjects were 44 orphans exposed once or twice to consecutive outbreaks of gastroenteritis due to type 3 rotavirus in an orphanage in Sapporo. Sera were collected throughout these outbreaks and the serum levels of neutralising antibodies against four different serotypes of group A human rotavirus were measured before and after the outbreaks. Protection against rotavirus gastroenteritis seemed to be serotype specific and to be related to levels of antibody against homotypic virus. A neutralising antibody level of 1/128 or greater seemed to be protective. The protective effect was of short duration, which was probably the explanation for recurrent attacks of gastroenteritis due to a rotavirus of the same serotype. Seroconversions or concomitant antibody responses to type 1 or 4 rotavirus in most children with type 3 rotavirus infection suggested that immunity to heterotypic virus can be induced by a rotavirus vaccine.

Antibodies, Heterophile↗
PubMed1986

Reactivity patterns to human rotavirus strains of a monoclonal antibody against VP2, a component of the inner capsid of rotavirus. Brief report.

A non-neutralizing monoclonal antibody (YO-60) against human rotavirus was found to be directed to VP2 (90,000-dalton protein), one of the two major components of the inner capsid. The reactivity patterns of the YO-60 antibody were very similar, though not identical, to those of subgroup II-specific YO-5 monoclonal antibody directed to VP6 (42,000-dalton protein), the other major component of the inner capsid. These results indicated the possible presence of a subgroup-specific antigen on VP2 in addition to the one on VP6.

Antibodies, Monoclonal↗
PubMed1986

Genetic reassortment between two human rotaviruses having different serotype and subgroup specificities.

Two reassortant viruses were selected from a mixed infection of MA104 cells with human rotavirus strains Wa (serotype 1-subgroup II) and HN126 (serotype 2-subgroup I). Antigenic characterization and genotype analysis by polyacrylamide gel electrophoresis revealed that they were reassortants with novel antigenic compositions, i.e. serotype 1-subgroup I (C116) and serotype 2-subgroup II (C15). Furthermore one of them, C15, was considered to have a mosaic antigenicity defined by two serotype-specific antigens, namely the serotype 1-specific VP3 antigen and the serotype 2-specific VP7 antigen. Although this reassortant was shown to be a serotype 2 virus on the basis of its preferential reactivity in neutralization reactions with serotype 2 antiserum, unexpectedly the antiserum prepared against C15 equally neutralized both serotype 1 and serotype 2 strains.

Antigens, Viral↗
PubMed1986

Use of microplate cell culture and enzyme immunoassay in titration of serum neutralizing antibody against Hochi strain of serotype 4 human rotavirus.

The tube neutralization test read by enzyme immunoassay developed by Wyatt et al. (1983) for serotype determination of human rotavirus was modified so as to use stationary cultures of MA104 cells in a microtiter plate instead of roller tube cultures. Sera obtained from different age groups were titrated for neutralizing antibody against serotype 4 human rotavirus Hochi strain by this test and the results were compared with those obtained by the plaque neutralization test. There was a good correlation between the titers obtained by the two tests and the age distribution pattern of serotype 4 neutralizing antibody was similar to those of serotype 1 and 3 antibodies previously reported.

Antibodies, Viral↗
PubMed1985

Preparation and characterization of neutralizing monoclonal antibodies with different reactivity patterns to human rotaviruses.

By employing three strains of cultivable human rotaviruses with different serotype specificity as immunizing antigens, we prepared 11 hybridomas which secreted neutralizing monoclonal antibodies against human rotaviruses. In neutralization tests with four strains of serotype 1, and three each of serotypes 2 and 3, the monoclonal antibodies showed different reactivity patterns: seven monoclonal antibodies reacted specifically with all strains of either serotype 1, 2 or 3 human rotavirus, but two showed strain-specific reactions; the remaining two were commonly reactive to various human rotavirus strains from each serotype but not to two non-human rotaviruses. By immunoprecipitation analysis, it was found that four serotype 2-specific and two commonly reactive antibodies were directed to VP3 (82000 mol. wt. protein) on the outer shell of the virus particles.

Antibodies, Monoclonal↗
PubMed1985

Transforming potency of flavoring agents in Chinese hamster cells.

To investigate the transforming potency of flavoring agents, Chinese hamster (CH) B241 cells were treated with various concentrations of flavoring agents for 24 hours during their exponential growth period. Surviving cells were cultivated for generations without the agents to observe the appearance of growth properties characteristic of transformed cells. Highly increased rates of structural and numerical chromosome abnormalities were seen in the cells treated with a high concentration of the agents, especially with allylisothiocyanate (AITC) or tr-cinnamic aldehyde (CA). During subsequent passages of the treated cells, the survivors from treatment with sublethal doses of AITC or CA showed a significant increase in cloning efficiency in soft agar medium, especially after passage in soft agar. Also noted was an increase in saturation density in monolayer culture, though a significant increase in plating efficiency at low serum level was not observed. These characteristic changes in AITC- or CA-treated cells were associated with the significant increase in frequencies of cells containing almost 3n to 4n chromosomes.

Animals↗