Morihiro Morita

Application of RT‐PCR Designed from the Sequence of the Local SRSV Strain to the Screening in Viral Gastroenteritis Outbreaks

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/SM82 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 pre‐amplification 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.

Isolation and Characterization of Mumps Virus Strains in a Mumps Outbreak with a High Incidence of Aseptic Meningitis

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 BamHI restriction cleavage site of the P gene, like the Urabe strain (Yamada, A. et al, Vaccine 8: 553‐557). 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.

Cloning and Characterization of the Genomic RNA Sequence of the Mumps Virus Strain Associated with a High Incidence of Aseptic Meningitis

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.

Molecular identification of two distinct hemagglutinin types of measles virus by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP)

Contemporary isolates of measles virus, characterized by their inability to hemagglutinate, have been shown to possess a hemagglutinin type distinct from that of classical strains such as the Edmonston strain in that there is a new glycosylation site at amino acid residue 416. This change abolishes a Sau3Al site that is found in the corresponding position of the hemagglutination-positive classical strains. This molecular information prompted us to develop a restriction fragment length polymorphism (RFLP) assay that is capable of distinguishing these two distinct hemagglutinin types. The assay consists of the amplification of a 349-bp segment of the hemagglutinin gene by reverse transcription followed by the polymerase chain reaction and Sau3Al digestion of this amplification product. The resulting two distinct RFLP patterns identified the hemagglutinin types with regard to the presence or absence of the potential new glycosylation site. This assay was applied to determine the relative frequencies over a 28-year period of these two hemagglutinin types present in the archival acute serum specimens taken from patients with measles. This study revealed that strains carrying the classical hemagglutinin type predominated until the early 1980s when it became completely replaced with strains possessing the contemporary hemagglutinin type. Because of its direct applicability to the clinical specimens avoiding selection bias during cell-culture adaptation, this assay provides a valuable asset in both clinical laboratory and epidemiological settings.

Cloning and characterization of the cDNA encoding the HA protein of a hemagglutination-defective measles virus strain

AbstractcDNA 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 Edmonstron 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 NaDodSO4PAGE 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.

Virological and epidemiological studies on an outbreak of aseptic meningitis caused by echovirus 4 in northern Japan in 1964

During the summer and autumn of 1964, echovirus 4 was found widely distributed in northern Japan; this virus was associated with localized outbreaks of iaseptic meningitis in many regions. The evidence that the echovirus was aetio-logically related to aseptic meningitis was shown by the virus isolation from cerebrospinal fluids and faeces and the serodiagnosis of paired sera in many patients: Age-specific sero-immunity and morbidity rate suggested that echovirus 4 had spread extensively in about 1954. The strains of echovirus 4 isolated in the 1964; epidemic were similar to the prototype strain, Pesascek, in biological characteristics.

水系感染集団事例から分離された毒素原性大腸菌および腸管集合性大腸菌耐熱性エンテロトキシン (EAST-1) 遺伝子保有大腸菌の性状

A water-borne outbreak occurred in A Town in Akita prefecture on March 1995. Enterotoxigenic Escherichia coli (ETEC) strains were isolated from 6 of 13 feces of patients with food poisoning disease and from 1 of 4 drinking water samples. In addition, E. coli strains harboring Enteroaggregative E. coli (EAggEC) heat-stable enterotoxin-1 (EAST-1) gene were isolated from 5 of 13 patients feces and 1 feces sample obtained from the septic tank. Both of the E. coli strains were isolated from the 3 patients feces, suggesting that this outbreak was a mixed infectious case. All of the ETEC strains possessed both heat-stable enterotoxin (ST) and EAST-1 genes and their serotype was O148:H28. The EAST-1 gene was detected on a ca. 80 kb plasmid by a southern blot analysis using EAST-1 DNA probe in the 5 of 7 ETEC strains. The southern blot analysis suggested that the location of the EAST-1 gene was genome in the rest of the 2 ETEC strains. A southern blot analysis using ST DNA probe also suggested that the location of the ST gene was genome in all of the ETEC strains. On the other hand, all of the 6 E. coli strains harboring EAST-1 gene could not be serotyped with commercially available OH sera. The location of the EAST-1 gene in all of the isolates was suggested to be genome by the southern blot analysis. All of the isolates lacked aggA gene which has been demonstrated to be involved in expression of aggregative adherence phenotype in EAggEC, suggesting that the EAST-1 gene-harboring strains isolated in this case were distinct from EAggEC. These results indicated that the EAST-1 gene was also harbored by E. coli strains distinct from EAggEC. In addition, a possibility was also suggested that the EAST-1 gene might be a transposon, as well as ST gene. Further study should be conducted in order to elucidate the significance of EAST-1 as a vilurence factor of diarrheagenic E. coli.

The Passive Hemagglutination Inhibition Test for Detection of Staphylococcal Enterotoxin B with Sensitized and Lyophilized Red Blood Cells

A passive hemagglutination inhibition (PHAI) test for the detection of Staphylococcal enterotoxin B (Ent. B) using tanned sheep erythrocytes has already been reported by Morse and Mah (5). This test has the advantages of sensitivity and quickness. However, the stability of the sensitized erythrocytes was not necessarily adequate to obtain reproducible results. This paper describes a new procedure to sensitize erythrocytes with enterotoxin B which resulted in obtaining stable erythrocytes under lyophilized conditions. Ent. B was prepared from the culture supernatant of Staphylococcus aureus strain C-243 as described by Schantz et al (7). The strain was kindly provided by Dr. H. Zen-Yoji, Tokyo Metropolitan Research Laboratory of Public Health. In brief, the organisms subcultured in heart infusion broth for 18 hr at 37 C were inoculated into a liquid medium (pH 6.8) consisting of 3% polypepton, 3% lactalbumin hydrolysate, 0.001 % niacin and 0.0005% thiamine hydrochloride

A passive hemagglutination test for detection of antibodies against M antigen of group A type 12 streptococci in human sera.

In seroepidemiological surveys on group A streptococci (S. pyogems), typespecific T antigen found on the cell surface of the organisms has been considered one of the immunological markers (8, 9). The antigen, however, plays no role in the virulence of the organisms. On the other hand, the acid-extractable M protein, that is also type specific and located on the cell surface, is known to be an important factor in the pathogenicity of these organisms (7). Since antibo:ly raised against M protein protects the host from infection by the same type of streptococcus (3, 5), the anti-M antibody may play an important role in seroepidemiological surveys of the organisms. To detect anti-M antibody in human sera, the passive hemagglutination (PHA) test, using tannic acid-treated erythrocytes, has been employed by some investigators (10-13). In general, the high sensitivity of the PHA test has been widely recognized. To expect consistent results of the test, however, the quality of the antigen used and reproducibility of the erythrocyte preparation are critical. From that point of view, the known procedures for purification of M antigen (4, 6, 7) and Boydens technique for preparation of sensitized erythrocytes (I) are not fully satisfactory. The present paper deals with the development of a new PHA test to detect anti-M antibody in human sera, using M antigen purified from a crude extract of type I 2 streptococci by a simple procedure. An M-protein-rich strain of group A type I 2 streptococcus, kindly provided by Dr. H. Kodama (Toyama Prefectural Institute of Public Health), subcultured in Todd-Hewitt broth (Difco) for I8 hr at 37 C, was inoculated into Todd-Hewitt broth supplemented with 10 g of glucose, 8 g of NaHC03, and I g of Na2HP04 per liter (2) and incubated for 30 hr at 37 C. The cells were centrifuged, washed twice with phosphate buffered saline (PBS pH 7.2), and resuspended in PBS (up to 25%). To the cell suspension, 1.0 N HCl was added until the pH reached 2.0 and the mixture was heated in a water bath for 10 min at 95 C. The suspension was then cooled, neutralized with 1.0 N NaOH, and centrifuged at 10,000 xg for 60 min. The supernatant containing crude M protein was dialyzed against PBS