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AIMS: To investigate the genetic differences among the strains of adenovirus type 8 (Ad8) circulating in Hiroshima city, Japan, and to study their circulation pattern. METHODS: One hundred and twenty nine strains of adenovirus type 8 (Ad8) were isolated in Hiroshima City over a 15 year period (1983-97) from patients with keratoconjunctivitis, and analysed with six restriction enzymes-BamHI, HindIII, PstI, SacI, SalI, and SmaI-to investigate possible relations among the isolates and their genetic variability. Seven hypervariable regions of the hexon gene that carry the type specific epitope were also sequenced to investigate the variation among the genome types. RESULTS: Restriction endonuclease analyses yielded three known genome types (Ad8A, 13 samples; Ad8B, seven samples; and Ad8E, 35 samples) and a novel genome type (Ad8I, 74 samples). Ad8A, Ad8B, and Ad8E were closely related, with 96% homology, whereas Ad8I had only 71% homology. Ad8A, Ad8B, and Ad8E shared 91.8% and 96.4% homology with regard to their amino acid and nucleotide sequences, respectively, with the isolate 1127 (accession no X74663). However, when compared with Ad8A, Ad8B, Ad8E, and isolate 1127, Ad8I shared only 62.7% and 69.9% homology with regard to amino acid and nucleotide sequences, respectively. Ad8A, Ad8B, and Ad8E had a unique 31 amino acid deletion in the hypervariable region 1 of the hexon gene, whereas Ad8I had a 33 residue deletion. The Ad8E strain that circulated from 1984 to 1995 was stable among the study population. Ad8I was isolated from an outbreak of epidemic keratoconjunctivitis in 1995 and was also isolated from sporadic cases until 1997. CONCLUSIONS: These results confirmed that genetic variability occurs in Ad8 in the microenvironment and revealed the emergence of a new genome type (Ad8I).

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Nucleotide and deduced amino acid sequences of the outer capsid proteins VP4 and VP7 of a murine rotavirus strain (YR-1) isolated in Japan were determined. Comparisons of the VP7 amino acid sequence of YR-1 with other murine rotavirus strains (EB, EW, EC, EL and EHP) (1) showed that YR-1 was highly homologous to EB, EW, EC, EL and EHP. Moreover, YR-1 was more closely related to strains representing G3 than to any other G type. Analysis of the VP4 amino acid sequence revealed that YR-1 was highly homologous to EB, EW, EC and EL [tentatively P17 (1)], and more closely related to EHP [tentatively P18 (1)] than to any other P type. Enzyme immunoassay with monoclonal antibodies against G types (KU-4 and BH49 for G1, S2-2G10 and BW36 for G2, YO-1E2 and BC5 for G3; and ST-2G7 and BE18 for G4) and against a P type (YO-1S3, KU-12H and YO-2C2 for P8) showed no reactivity. These results indicate that YR-1 is highly homologous to EB, EW, EC and EL.

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We report herein the case of a 46-year-old woman on hemodialysis (HD) who developed recurrent renal hyperparathyroidism induced by lung metastasis from parathyroid carcinoma. The patient had been commenced on HD for chronic renal failure about 20 years earlier and had undergone a parathyroidectomy for advanced renal hyperparathyroidism 8 years later. After the initial operation, further explorations of the neck were performed due to recurrence, despite which the hyperparathyroidism persisted and she was finally referred to our department. The appearance of multiple coinlike lesions on a chest X-ray and computed tomography led to the diagnosis of recurrent hyperparathyroidism induced by lung metastasis from parathyroid carcinoma. A pulmonary wedge resection was performed and the metastatic parathyroid nodules were removed. Of the several hypotheses about the etiology of parathyroid carcinoma in HD patients, it is most likely that the parathyroid hyperplasia induced by chronic renal failure develops into carcinoma. Even in renal hyperparathyroidism, we should bear in mind the possibility that metastatic parathyroid carcinoma is a possible source of excess parathyroid hormone secretion at recurrence.

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Serotyping of human rotavirus in the Tokyo area was conducted from 1990 to 1993 by enzyme immunoassay with monoclonal antibodies (EIA-MAbs) against VP7 and by reverse transcription and polymerase chain reaction (RT-PCR) amplification of the VP4 and VP7 genes. The results by EIA-MAbs were very similar to those obtained by RT-PCR. Evidence of intraserotypic variations was suggested because strains of undetermined serotypes were detected by either EIA-MAb or RT-PCR. This kind of study is required for vaccine development.

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Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on 12 human isolates of serotype 1 of rotavirus in Japan and China. They were examined for genetic variations among serotype 1 isolates. Comparative studies of their nucleotide and deduced amino acid sequences between the 12 isolates and the Wa strain revealed an overall homology of more than 92 and 96%, respectively. Higher degrees of homologies were observed between Wa and 2 strains (K1 and K2) in Tokyo, 1979-1980, than between Wa and recent isolated strains in Tokyo and in China. In our isolates, a total of 16 amino acid residues frequently converted to another amino acid. Six amino acid residues belonging to the major neutralizing epitope regions (B, D, and E in this communication) frequently converted. From these data three subtypes (subtypes A, B, and intermediate) were suggested to be divided. Whether these differences are an important mechanism in the epidemiology of rotaviruses requires further investigation.

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Two series of enzyme immunoassays with monoclonal antibodies produced in two laboratories (A and B) were compared in use for serotyping of human rotavirus in stool samples collected in Japan between 1988 and 1991 from patients with gastroenteritis. Of 358 samples, 222 were determined to be the same serotype, while 61 samples could not be serotyped by either assay. A hundred and one and 92 samples were not serotyped by the A and B antibodies, respectively. We believe that the A and B monoclonal antibodies are useful clinically for serotyping rotaviruses at the present time.

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A seroepidemiological study of rotavirus was conducted in the northern part of Tokyo from 1990 to 1992 by using reverse transcription-polymerase chain reaction (RT-PCR). G1 and G3 types were detected in the winter between 1990 and 1991, however G1 type was appeared mainly in the winter between 1991 and 1992. G3 type was observed as the main type during the winter in the 10 year survey in the Tokyo area for the first time. RT-PCR was useful in the seroepidemiological studies of rotavirus infection.

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Direct rotavirus serotyping (VP7, G type) in stool specimens was carried out by reverse transcription and polymerase chain reaction amplification (RT-PCR) and compared to serotyping by enzyme immunoassay with monoclonal antibodies (EIA-MAb). The methods used for double-stranded (ds) RNA extraction, RT-PCR amplification, and the primers used were modified from previous reports [Gouvea et al.: Journal of Clinical Microbiology 29:519-523, 1990; Gentsch et al.: Journal of Clinical Microbiology, 1992]. For samples that were positive by both methods, the serotypes obtained were identical, however RT-PCR typing was found to be considerably more sensitive (70.4% samples serotyped) than EIA-MAb (35.6% of samples serotyped). The overall sensitivities for detection of rotavirus in stool samples by latex agglutination, enzyme immunoassay, electron microscopy, polyacrylamide gel electrophoresis, and RT-PCR were essentially the same. The results confirm that RT-PCR typing (genotyping) is extremely valuable for G typing of samples which cannot be typed by EIA-MAb. We also developed a PCR confirmation technique for serotypes 1, 2, and 4.

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Epigallocatechin gallate from green tea and theaflavin digallate from black tea inhibited infections of cultured rhesus monkey kidney MA 104 cells with rotaviruses and enteroviruses. Their antiviral effects were maximally induced when directly added to virus, and their pre- and post-treatment of the cells produced much weak antiviral activity. Antiviral activity of the extracts therefore seems to be attributable to interference with virus adsorption.

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Succeeding the previous report on antivirus activity of non immunized bovine colostrum immunoglobulin, we studied the bacteriostatic activity of the immunoglobulin, lactoferrin and lactoferrin Fe by using the automated antimicrobial susceptibility testing system. We have presented the representative positive and negative reactions. Then, we have reported that the immunoglobulin was effective in 10 of a total of 20 species which we examined. In some species, the lactoferrin was slightly more effective than the immunoglobulin was. These results indicated that the immunoglobulin, lactoferrin and lactoferrin Fe were useful for the bacteriostatic reaction.

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Bovine colostrum whey and immunoglobulins were prepared. Their characteristics and anti-viral activities were studied:IgG, IgA and IgM were found in bovine colostrum. Most IgG was polymerized. Although neutralization activities against bovine, simian and human rotaviruses existed, anti-human adenovirus antibody was not found. Effects on prophylaxis and treatment for rotavirus gastroenteritis were expected.

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Epidemiology of rotavirus infection was studied from 1981 to 1988 mainly in three hospitals around Tokyo area. Major serotypes of rotaviruses in the three places were different from those in two hospitals around Kansai area in Japan (Ref. 6, 13), while, major serotypes were same among three hospitals. Both of serotypes 1 and 4 in group A were mostly found around Tokyo area. Frequencies of type 2, 3, and 9 in group A were low, although the frequencies were various among periods. Detail examinations of rotavirus RNA electropherotypes showed the results as follows; different electropherotypes were found during one winter season and at one hospital, the identical electropherotype was found cross a year and cross a hospital. We could not find the identical electropherotype which belong to two serotypes so far. Seven group C rotaviruses were found since 1987 in three hospitals. It would be important to examine RNA electropherotypes and serotypes for long period not only for epidemiological studies but also for development of vaccine.

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Four human group C rotaviruses were detected in Tokyo in 1987 and 1988 during a survey over 7 years. Among the four rotaviruses, two electrophoretic patterns were indicated by polyacrylamide gel electrophoretic (PAGE) analyses. Clinical symptoms, signs, family history, and patients' ages varied. Group C rotaviruses were found also in other parts of Japan in 1988. It was suspected that group C rotaviruses would continue to spread throughout Japan within the near future.

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We investigated the antigenic and genetic characters of one of two human rotavirus strains 69M and 57M isolated in Indonesia, both of which showed a "super-short" RNA electrophoretic pattern (A. Hasegawa et al., Microbiol. Immunol. 28, 719-722, 1984). By an enzyme-linked immunosorbent assay with subgroup-specific monoclonal antibodies, one virus, strain 57M, was found to have subgroup II antigenicity. The cross-reaction of this strain, in a plaque neutralization test, with a serotype 4 strain was high whereas that of strain 69M was low. When radiolabeled RNA probes prepared from this virus were hybridized with RNAs from reference strains of different serotypes, treated with S-1 nuclease and then subjected to polyacrylamide gel electrophoresis, we found that (i) RNA segment 10 (the super-short segment) hybridized with that of another super-short pattern virus, strain 69M; (ii) segment 7, coding for a neutralization antigen, hybridized with that of the serotype 4 virus; (iii) segment 6, coding for a major inner-shell protein, hybridized with that of a serotype 1 virus; and (iv), some other segments hybridized with those of the reference viruses of serotypes other than 2 and 3. We suspect that this strain is possibly a naturally-occurring reassortant virus whose genetic segments are derived from different human rotaviruses.

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We investigated genetic and serological characteristics of a human rotavirus isolate from Indonesia which had a "super short" RNA electrophoretic pattern (A. Hasegawa, S. Inouye, S. Matsuno, K. Yamaoka, R. Eko, and W. Suharyono, Microbiol. Immunol. 28:719-722, 1984). This virus, strain 69M, was found by RNA-RNA hybridization to have a low degree of homology with the representative strains of all four human serotypes. Furthermore, it could not be classified by neutralization analysis into any of these serotypes. Therefore, this virus might belong to a new serotype.

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Enterovirus type 70 (EV70) agglutinated human 'O' erythrocytes at 4 degrees C as well as 22 degrees C, but visible agglutination was lost when warmed at 37 degrees C although the virus remained attached to the surface of the erythrocyte. The receptor sites for the virus were neuraminidase-sensitive. A direct involvement of sialic acid on the cell surface in virus-cell interaction was confirmed by the fact that the presence of fetuin or free N-acetylneuraminic acid inhibited the haemagglutinating activity of EV70. Similar numbers of virus particles were required for 1 haemagglutinating unit (HAU) of EV70 and 1 HAU of mengovirus, whereas 2.6-fold or more of virus particles of echovirus type 7 and type 11 gave the same activity. On the other hand, the number of receptor sites on the cell surface for EV70 was found to be sevenfold more than for mengovirus. Therefore, the erythrocyte receptor for EV70 is different from that for common enteroviruses and similar, though not identical, to the cardiovirus receptor. However, serological tests such as neutralization, complement fixation or haemagglutination inhibition did not reveal any common antigen between EV70 and cardiovirus.

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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.

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Dextran sulfate aggregates several enteroviruses depending not only on the pH, the ionic strength of the medium, but also on the protein content of the fluids and on strain specificities of the viruses. The aggregation effect was measured by filtration experiments, by sedimentation in the ultracentrifuge and by electron microscopy. The well known inhibiting effect of dextran sulfate on plaque formation may be due to its aggregating effect: A very strong inhibition of the release of matured virions from the infected cells is observed in medium containing dextran sulfate, whereas the adsorption process is inhibited much less compared with PBS controls. The maximal effect on virus aggregation, plaque size and virus release is observed at the same concentration of dextran sulfate.

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