In 1993, mumps with a high incidence of aseptic meningitis became prevalent in Akita prefecture, Japan. Three mumps virus isolates obtained from the nonvaccine-associated cases lacked the B amHI restriction cleavage site of the P gene, like the Urabe strain (Yamada, A. et al,. However, four additional nucleotide substitutions were found in the determined region of 157 bp. Fourteen of 19 cases from which mumps virus showing the Urabe-like RFLP profile was detected were complicated with symptomatic meningitis, whereas there were only four cases of meningitis among 23 individuals infected with the wild type showing no Urabe-like RFLP profile (non-"Urabe-like" wild-type). The incidence of meningitis was over 70% among patients infected with the "Urabe-like" wild-type virus. The "Urabe-like" wild-type disappeared after February 1994 in the epidemic area and was replaced by the non-"Urabe-like" wild-type. Patients infected with the "Urabe-like" wild-type lived in a closed colony, in which there were two instances of transmission between siblings. Thus this outbreak was transient and narrowly localized.

To determine the significance of poultry and bovine as infectious sources of Campylobacter jejuni in Japan, the serotype distribution and pulsed-field gel electrophoresis (PFGE) patterns of poultry and bovine isolates were compared with those of isolates from patients with diarrhea in Akita (Japan). Serotypes O:2 and O:4-complex were common in human, poultry, and bovine isolates, and serotype O:23,36,53 was common in human and bovine isolates. SmaI PFGE patterns of isolates belonging to these serotypes were generated. Eight PFGE patterns were shared by poultry and human isolates and three patterns were shared by human and bovine isolates. Further analysis of the isolates having the same SmaI PFGE pattern by KpnI PFGE confirmed that four patterns and two patterns were still shared by poultry and human isolates, and bovine and human isolates, respectively. Thus, serotypic and genotypic data indicated a possible link between sporadic human campylobacteriosis and C. jejuni from retail poultry and bovine bile and feces, suggesting that bovine serves as an infectious source of C. jejuni in Japan, as is observed in other countries.

Four bla VIM-2 gene-harboring Pseudomonas aeruginosa strains were identified. These strains possessed a class 1 integron harboring ORF1, bla VIM-2 , and aacA4 gene cassettes. The transposon-mediated horizontal spread of the bla VIM-2 gene among these strains was suggested, which increases the threat that the bla VIM-2 gene will disseminate among diverse genera of bacteria.

The Yuri strain of small round structured virus (SRSV) was cloned from a fecal specimen in which virus particles were observed by electron microscopy. The most common RT-PCR protocol in Japan, however, using 35/36 and NV81/NV82/SIV182 nested primer pairs, could not detect the SRSV genome in this specimen. Nucleotide and amino acid sequence analysis revealed that the Yuri strain is genetically close to the genotype II of SRSV. A novel procedure using primer sets designed from the nucleotide sequence of the Yuri strain was applied to the screening of 119 stool samples obtained from subjects with sporadic diarrhea and 46 samples obtained during seven foodborne gastroenteritis outbreaks. Using this novel procedure, PCR bands were detected in 44% and 52% of the samples, respectively. These detection rates were approximately twice those obtained with the 35/36 and NV81/NV82/SM82 nested primers. In particular, more than 40% of positive samples could be detected by using only the Yuri primer sets. Furthermore, three improvements were made in the RNA preparation, cDNA synthesis, and amplification steps to save materials and time. The background, or extra bands, in the amplification reaction resulting from DNA in the fecal specimens was completely removed by DNase I treatment just before cDNA synthesis. Random nonamers were used as universal primers in the reverse transcription. No difference in sensitivity or specificity was noted in the final results when either random nonamers or specific primers were used. The use of a preamplification step under low stringent conditions before standard amplification under highly stringent conditions compensated for any mismatched bases in the primers with respect to the target sequences. Thus our novel procedure using Yuri primer sets may be useful for the screening of SRSV in the recent SRSV outbreaks in Japan.

cDNA clones of the mumps virus wild-type strain, associated with a high incidence of aseptic meningitis (ODATE-1 strain), were isolated and analyzed from genomic nucleotide position 22 to 8520 containing the NP, P, M, F, SH and HN protein coding region. The ODATE-1 strain exhibited a RFLP profile identical to that of the Urabe vaccine strain in spite of the fact that the virus was isolated from non-vaccinated cases. However, a comparison of nucleotide and amino acid sequences among the ODATE-1 strain, Urabe strain and Miyahara strain revealed that the ODATE-1 strain was not related to the Urabe strain. The mumps virus (MV) is one of the medically important paramyxoviruses within the paramyxovirus family. In 1993, mumps associated with a high incidence of aseptic meningitis became prevalent in Akita Prefecture, Japan. We have previously reported that two types of MV distinguishable by their restriction fragment length polymorphism (RFLP) profiles were isolated in the outbreak (5). One type (the "Urabe-like" wild-type) lacked the B amHI restriction cleavage site of the P gene, like the Urabe vaccine strain (12), in spite of the fact that the virus was isolated from non-vaccinated cases. Moreover, the incidence of meningitis among patients infected with the "Urabe-like" wild-type strain was over 70%, compared with 17% in those infected with the other "non -Urabe-like" wild-type strain (5) . A partial nucleotide sequence analysis revealed that the "Urabe-like" wildtype strains (ODATE strains) were not identical to the Urabe vaccine strain. Although the possibility that the Urabe strain had reverted to the wild-type cannot be completely ruled out, no evidence of this was obtained from the result of our previous study. This molecular information prompted us to analyze other genomic regions of the ODATE strain.The MV is composed of unsegmented negative-sense

cDNA clones corresponding to the mRNA for the hemagglutinin of the hemagglutination-defective strain AK-1 of measles virus were isolated and characterized. Compared with the prototype Edmonston strain, 60 nucleotide substitutions that resulted in 18 amino acid changes were detected. An additional potential N-linked glycosylation site was added by point mutation, which was supported by the observation that the hemagglutinin of the AK-1 strain was stained more heavily after NaDodSO4-PAGE and periodic acid-Schiff (PAS) staining than the Edmonston strain. Computer-assisted analysis revealed that three reverse turns in the secondary structure had disappeared in the hemagglutinin of the AK-1 strain. Moreover, one of these structural changes occurred in the closely glycosylated region at amino acid residues 168-240, which appeared to be a biologically important functional domain. The isoelectric point calculated from the predicted amino acid sequence became about 1 pH unit more basic in the AK-1 strain than the Edmonston strain. This present study is the first sequence analysis of the hemagglutinin gene in a hemagglutination-defective strain of the measles virus.

The class 1 integrons are genetic elements capable of integrating gene cassettes by a site-specific recombination mechanism (1). Gene cassettes are mobile units composed of a gene, most often an antibiotic resistance gene, and a recombination site, the 59-base element (1). We have previously characterized a class 1 integron containing a VIM-2-type metallo-␤-lactamase gene, bla VIM-2 , an aacA4 gene, and an unknown function gene, designated as ORF1, which was identified in a Pseudomonas aeruginosa clinical isolate strain M␤-7 (6). Partridge and Hall (5) recently searched using the predicted sequences of the proteins encoded by gene cassette-encoded open reading frames and revealed that two proteins encoded by ORF1 and orf "i" (2) genes showed a similarity with the amino acid sequence, along with the presence of a number of conserved key amino acids, of known fosfomycin resistance determinants including FosA, FosB, PA1129, lmo1702, and mlr3345. Based on these search results, they proposed that both ORF1 and orf "i" genes are likely to confer fosfomycin resistance. To prove this hypothesis, we cloned the ORF1 gene and constructed an Escherichia coli transformant to examine its antibiotic resistance phenotype.The ORF1 gene was amplified from P. aeruginosa strain M␤-7 by PCR using primers ORF1 Exp5 EcoRI (5Ј-TCG GAA TTC AAT GAT TAC CGG CAT CAA TCA C-3Ј) and ORF1 Exp3 HindIII (5Ј-TTA AAG CTT CGT CAG CTC CAC ACC AGC CCC TT-3Ј). These primers were designed to clone the ORF1 gene to be in frame with the lacZ gene on the cloning vector pBCSKϩ to construct transformant able to express the ORF1 fusion protein. Since these primers prime at the beginning and the end of the ORF1 coding region, respectively, all regulatory signals from the original P. aeruginosa gene were eliminated. The amplified fragment containing the ORF1 gene was digested with EcoRI and HindIII and cloned into pBCSKϩ to form the recombinant plasmid, ORF1: pBCSKϩ. E. coli DH5 was transformed with the ORF1: pBCSKϩ plasmid to form E. coli DH5 ORF1:pBCSKϩ. The transformants were selected on L agar plates containing 30 g/ml chloramphenicol and 20 mM IPTG (isopropyl-␤-D-thiogalactopyranoside). Antibiotic resistance phenotypes of the E. coli DH5 ORF1:pBCSKϩ, P. aeruginosa strain M␤-7, and E. coli DH5 containing pBCSKϩ (E. coli DH5 pBCSKϩ) were determined by the broth microdilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (3, 4) using commercially available plates, Dryplate "Eiken" DP-21 and DP-25 (Eiken Kagaku Co., Tokyo, Japan).P. aeruginosa strain M␤-7 and E. coli DH5 ORF1:pBCSKϩ showed a Ͼ32 g/ml MIC of fosfomycin, while the control strain E. coli DH5 pBCSKϩ was sensitive to this antibiotic. These results prove the hypothesis proposed by Partridge and Hall (5) that the ORF1 gene is the novel fosfomycin resistance determinant. It is important to clarify prevalence of this novel fosfomycin resistance determinant.

Table 1 Drugs for MIC determination Table 2 MIC distribution in human diarrhea-derived C. jejuni strains