[Experience with invasive thymoma presenting pleural dissemination].

A 61-year-old man was admitted to Showa University Hospital because of a myasthenia gravis. Chest computed tomography revealed a mediastinal invasive tumor. During surgery, invasion to the pericardium and dissemination on the left visceral pleura and the left diaphragm were observed. Extended thymo-thymectomy and partial resection of the pericardium, left lung, and diaphragm were performed. Incomplete resection was achieved because of the dissemination on the diaphragm. Chemotherapy using ADOC and radiotherapy for mediastinum and left diaphragm were done. Four years after surgery, neither recurrence of the tumor nor myasthenia gravis was observed.

[A case of chronic methyl bromide intoxication showing symmetrical lesions in the basal ganglia and brain stem on magnetic resonance imaging].

A 30-year-old man developed paresthesia in the feet, unstable gait and blepharoptosis subsequent to pharyngeal pain while working in a warehouse of imported fruit and vegetable fumigated with methyl bromide and cyanate. Neurological examination showed bilateral blepharoptosis, diplopia, ataxic gait and paresthesia below the inguinal region. The serum concentration of thiociane, a metabolite of cyanide, was within normal limits, but that of bromide was elevated (43.7 mg/l, normal: 3.7-8.6). Thus we diagnosed the patient as intoxication of methyl bromide. Magnetic resonance imaging of the brain (MRI) revealed bilateral symmetrical lesions of high signal intensity in the posterior region of the putamen, the subthalamic nuclei, the dorsal medulla oblongata corresponding to inferior cerebellar peduncles and vestibular nuclei, the inferior colliculi and the periaqueductal gray matter surrounding the oculomotor nucleus of the midbrain. As far as we know, this is the first report of MRI detected cerebral lesions in a case with intoxication of methyl bromide.

[Amnesia following herpes simplex encephalitis].

We describe three patients presenting themselves with amnesia following herpes simplex encephalitis. All three patients showed anterograde amnesia. Moreover, one patient showed retrograde amnesia extending about fifty years prior to the onset of the illness. Two patients revealed semantic memory disorder, disinhibitory behaviors and confabulations, which suggested the existence of frontal lobe dysfunction. MRI of all three patients disclosed uni- or bilateral temporal lesions. SPECT images showed decreased uptake not only in the temporal lobes but also in the frontal lobes. Amnesia following herpes simplex encephalitis has been thought to be caused by the temporal lobe disorder. However, we postulate that the frontal lobe dysfunction might contribute to the appearance of amnesia.

Characterization of human rotavirus genotype P[8]G5 from Brazil by probe-hybridization and sequence.

We report the molecular characterization of rotavirus genotype P[8]G5 strains found in fecal specimens collected in four different regions of Brazil, using digoxigenin(dig)-labeled oligonucleotide probes, sequence analysis, and RNA-RNA hybridization. The closest sequence relationships of the neutralization antigens of these strains were to the VP4 protein of P1A[8]G1 strain KU (93.3% identity in amino acids 11 to 282) and to the VP7 protein of G serotype 5 strain OSU (87.6% identity in amino acids 8 to 232). Based on VP7 sequence differences, we designed dig-probes that allowed us to discriminate porcine OSU-like strains from G5 strains isolated from Brazilian infants. The genetic relationships of two P[8]G5 isolates to other rotavirus genogroups were analyzed by RNA-RNA hybridization with [32P]-GTP probes representative of serotypes P1A[8]G1 (Wa), P[8]G3 (AU17), and P9[7]G5 (OSU). The Brazilian P[8]G5 strains showed sequence homology with genes of Wa-like and OSU-like strains, suggesting that these two strains were naturally occurring reassortants between members of the Wa and porcine rotavirus genogroups. The identification of these strains in diverse geographic areas of Brazil underscores their stability and demonstrates the emergence of clinically important rotavirus diarrhea strains by reassortment.

Distinctness of NSP1 gene of human rotavirus AU-1 from NSP1 gene of other human genogroups.

Human rotavirus strain AU-1 has a genomic RNA constellation distinct from that of either the Wa or DS-1 genogroup and thus, represents the third human rotavirus genogroup. Nucleotide sequence analysis of the non-structural protein 1 (NSP1) of AU-1 revealed that it was only 53-57% identical at the amino acid level with strains belonging to either the Wa or the DS-1 genogroup. This result confirmed and extended the earlier observation that the grouping based on the NSP1 sequence similarity is usually in good agreement with classification by genogroup. Phylogenetic analysis placed the AU-1 NSP1 gene on the bovine NSP1 branch, although more than 13% amino acid difference was observed.

Molecular characterization of a human group A rotavirus isolated from an adult with severe dehydrating diarrhea and its relationship to strains concurrently circulating among children.

BACKGROUND: While group A rotavirus is widely accepted as an important etiology of acute gastroenteritis in children, this agent rarely causes severe diarrhea in adults and, thus, is not considered by physicians to be an etiological agent for such diseases. OBJECTIVES: None of the reports describing such rare cases in adults has examined the causative strains genetically in detail. STUDY DESIGN: We determined the G type, the gene 4 genotype, the electropherotype, and the genomic RNA constellation (genogroup) of a group A rotavirus strain isolated from an adult with severe diarrhea. This patient, the first documented case of rotavirus diarrhea in adult in Japan, suffered from severe dehydrating diarrhea with 'rice-water' appearance, vomiting and little abdominal pain, presenting a clinical picture typical of cholera. RESULTS: Rotavirus antigen and genomic RNA were detected in the stool but other enteric pathogens including Vibrio cholerae responsible for the disease were not isolated. Molecular characterization revealed that the patient was infected with a strain of the DS-1 genogroup with G2 and gene 4 genotype 4 which was circulating among children during the same period. CONCLUSIONS: The result that the same group A rotavirus strain was isolated from children and an adult underlines the necessity of examining rotavirus in adults with acute diarrhea.

Recurrent circulation of single nonstructural gene substitution reassortants among human rotaviruses with a short RNA pattern.

To determine the relative frequency of intergenogroup reassortants of rotavirus in nature, we analyzed the genetic composition of 22 electrophoretically distinct stool isolates which accounted for 95.2% of stool rotaviruses with a short RNA pattern collected during 10 rotavirus seasons. These strains all showed subgroup I and G2 specificities, but two distinct hybridization patterns were observed when the probes prepared from Wa (a member of the Wa genogroup) and KUN (a member of the DS-1 genogroup) were used. Genomic RNAs from 10 strains (accounting for 64% of the field rotaviruses with short RNA pattern) hybridized exclusively to the KUN probe, and thus belonged to the DS-1 genogroup. On the other hand, genomic RNAs of the remaining 12 strains (accounting for 36% of the field rotaviruses with short RNA pattern) formed one hybrid band with the Wa probe and 10 hybrid bands with the KUN probe. Thus, they were single gene substitution intergenogroup reassortants between members of the Wa and DS-1 genogroups. They had a similar genetic constellation in that a gene segment encoding either NS35 or NS34 from a Wa-like strain was introduced into a DS-1-like genome background.

Serotype G6 human rotavirus sharing a conserved genetic constellation with natural reassortants between members of the bovine and AU-1 genogroups.

Serotype G6 human rotavirus PA151 was examined by RNA-RNA hybridization in comparison with another G6 strain PA169 and two naturally-occurring G3 reassortants PCP5 and MZ58. PA151 possessed three gene segments that formed hybrids with AU-1 and seven gene segments that formed hybrids with bovine rotavirus NCDV. PA151 also possessed eight, 10 and 10 gene segments that formed hybrids with genomic RNAs from PA169, PCP5, and MZ58, respectively. Thus, PA151 was an intergenogroup reassortant formed in nature between members of the bovine and AU-1 genogroups and it shared a genome constellation with PA169, PCP5, and MZ58. These results suggest that naturally-occurring intergenogroup reassortants possessing such a genome constellation were perpetuated in human populations.

Naturally occurring dual infection with human and bovine rotaviruses as suggested by the recovery of G1P8 and G1P5 rotaviruses from a single patient.

Culture adaptation of rotaviruses from an infant with severe diarrhea in Cincinnati, Ohio, yielded not only a virus with the original RNA electropherotype (CJN) but also rotaviruses with other electropherotypes, the most dominant of which was called CJN-M [Ward RL, Knowlton DR, Schiff GM, Hoshino Y, Greenberg HB (1988) in J Virol 62: 1543-1549]. RNA-RNA hybridization and sequencing studies indicated that CJN was a typical G1P8 human rotavirus while CJN-M was a G1P5 strain and contained four gene segments (including segment 4) of a bovine rotavirus. Thus, the infant was apparently dually infected with human and bovine rotaviruses.

The distribution of G (VP7) and P (VP4) serotypes among human rotaviruses recovered from Japanese children with diarrhea.

Both the G (VP7) and P (VP4) serotypes of human rotaviruses collected over a 10-year period from Japanese children with diarrhea were determined by recently-developed polymerase chain reaction-based typing assays. The combination of G1 and P8 was found in 65.2% and the combination of G2 and P4 was found in 15.2%. For the rest of the specimens, only a few other combinations occurred and their relative frequencies were less than 10%. The viruses carrying P9 were always associated with G3 as is the prototype strain AU-1.

Nucleotide sequence comparison of the VP8* gene of rotaviruses possessing the AU-1 gene 4 allele.

Of the five currently recognized alleles of the human rotavirus VP4 gene, the AU-1 allele has captured attention because of its possible non-human origin. The 5' 750 nucleotide region of the VP4 gene, encoding the VP8* fragment [amino acids (aa) 1 to 241] and the connecting peptide (aa 242 to 247), from 13 human and two feline rotavirus strains possessing the AU-1 allele was highly conserved both at the nucleotide sequence (93.8 to 99.7% identity) and amino acid level (95.5 to 100% identity) irrespective of the year and the place of isolation or of the host species from which these viruses were isolated. This is consistent with the hypothesis that the AU-1 allele of the VP4 gene has been maintained in both human and feline rotavirus gene pools.

Relative frequency of VP4 gene alleles among human rotaviruses recovered over a 10-year period (1982-1991) from Japanese children with diarrhea.

The relative frequencies of the Wa (corresponding to serotype P1A), DS-1(P1B), M37(P2), and AU-1(P3) alleles of the VP4 gene from rotaviruses collected from the stools of individuals in Japan between 1982 and 1991 were determined to be 83.1, 15.6, 0, and 1.3%, respectively, by a polymerase chain reaction-based typing assay.

Two-way cross-neutralization mediated by a shared P (VP4) serotype between bovine rotavirus strains with distinct G (VP7) serotypes.

Among bovine rotavirus strains, there are three G serotypes (G6, G8, and G10) and three P (VP4) serotypes (PB1, PB2, and PB3, which are defined on the basis of strains NCDV, UK, and B223, respectively). Plaque reduction neutralization assays with hyperimmune antisera disclosed two-way antigenic relationships of strain KN-4 with strain KK-3 (G10, PB3) as well as with strains NCDV (G6, PB1) and 0510 (G6, PB2). Neutralization assays with monoclonal antibodies specific for G6, G10, and PB3 revealed that KK-3 and KN-4 had the same P serotype (PB3) but that neither G6- nor G10-specific monoclonal antibody neutralized KN-4. Comparison of the VP7 gene sequence of KN-4 with those of other bovine rotavirus strains indicated that KN-4 was more similar to G6 bovine strains than to KK-3 and other G10 strains, suggesting that the G serotype of KN-4 was G6. From these results, we concluded that the two-way cross-neutralization between KN-4 and NCDV or 0510 was mediated by shared G6 serotype specificity, whereas the two-way cross-neutralization between KN-4 and KK-3 was mediated by shared P serotype specificity (PB3). Thus, KN-4 and KK-3 represent the first reported example of a two-way antigenic relationship mediated only by the P serotype. This article emphasizes the need for adopting a binary serotyping system and development of reagents which will enable classification of rotaviruses based on their G and P serotype specificities.

Thermal degradation of RNA-RNA hybrids during hybridization in solution.

Degradation of single-stranded RNA molecules at high temperatures was examined in relation to the kinetics of RNA-RNA hybridization in solution. Eleven species (ranging from 670 bases to 3300 bases) of single-stranded RNAs transcribed from rotavirus genomic RNAs degraded significantly after 16 h of incubation at 65 degrees C. The hybridization of these 11 RNA molecules to the corresponding genomic RNAs, however, was completed within 30 min of incubation. Partially homologous hybrids that were once formed at an early time of incubation gradually degraded in proportion to the length of incubation at 65 degrees C. Thus, the length of hybridization has a critical effect on the final hybridization results. Furthermore, thermal hydrolysis of single-stranded RNA molecules provides a plausible explanation why the percent of nucleotide sequence mismatch allowed to form a stable hybrid in the RNA-RNA hybridization assays for rotavirus genes is much less than that predicted by calculation.

Human rotavirus strain with unique VP4 neutralization epitopes as a result of natural reassortment between members of the AU-1 and Wa genogroups.

Human rotavirus strain K8, which possesses unique VP4 neutralization epitopes, was examined by RNA-RNA hybridization to determine its genogroup. While it possessed four gene segments that formed hybrids with strain Wa (a prototype of the Wa genogroup), strain K8 possessed seven gene segments, including gene segment 4, that formed hybrids with strain AU-1 (a prototype of the AU-1 genogroup) which has been shown to share a unique gene 4 allele with feline rotaviruses. These results suggest that strain K8 is an intergenogroup reassortant formed in nature between a member of the Wa genogroup and a member of the AU-1 genogroup.

Subgroup I serotype 3 human rotavirus strains with long RNA pattern as a result of naturally occurring reassortment between members of the bovine and AU-1 genogroups.

Two human rotavirus strains, PCP 5 and MZ 58, which possessed an unusual combination of subgroup (I), serotype (3) and RNA pattern (long) were examined by RNA-RNA hybridization to determine their genogroup. While these two strains did not belong to either the Wa or the DS-1 genogroup, PCP 5 and MZ 58 possessed seven gene segments that formed hybrids with bovine rotavirus strain NCDV and four gene segments that formed hybrids with human rotavirus strain AU-1. These results suggest that PCP 5 and MZ 58 were intergenogroup reassortants formed in nature between a member of the bovine rotavirus genogroup and a member of the AU-1 genogroup.

Human and bovine serotype G8 rotaviruses may be derived by reassortment.

The origin of, and relationship between human and bovine serotype G8 rotaviruses were investigated by genomic hybridisation. Radiolabelled mRNAs of human G8 rotaviruses 69M (isolated in Indonesia) and HAL1271 (isolated in Finland), and bovine rotaviruses KK3 (G10) and NCDV (G6), were used as probes. The products of liquid hybridisation between the probes and the genomic RNA of human and bovine rotaviruses, including bovine G8 rotavirus 678 (isolated in Scotland) and two other Finnish human G8 rotaviruses HAL1166 and HAL8590, were examined by separation in polyacrylamide gels. The genomes of Finnish human G8 rotaviruses were similar to those of bovine G6 and G10 rotaviruses. Neither Indonesian human G8 nor bovine G8 viruses had high levels of similarity to each other or to other bovine and human rotaviruses. Thus these three epidemiologically distinct G8 rotaviruses have different origins and may be derived by reassortment with rotaviruses of a third, as yet unknown, host species. The similarity between the Finnish isolates and the bovine isolate NCDV suggests that they have diverged recently and that these human G8 rotaviruses may be derived from a zoonotic infection, or alternatively, from the live rotavirus vaccine of bovine origin which has been used to vaccinate Finnish children.