Shozo Urasawa

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.

Sequential Passages of Human Rotavirus in MA‐104 Cells

Starting with a small amount of diarrheal feces containing human rotavirus (HRV), we succeeded in propagation of the virus using the roller culture technique with MA‐104 cells. Furthermore, we made a successful adaptation of HRV to a stationary culture and developed a plaque assay for the cell culture‐adapted viruses. The 3 culture‐adapted virus isolates, KU, YO, and 44 produced plaques (about 0.5–1.0 mm in diameter) under the overlay medium consisting of 0.6% purified agar, 3 μg of acetyl trypsin/ml and 50 μg of DEAE‐dex‐tran/ml. Subsequent plaque purification resulted in the formation of clear, larger plaques.

Detection of mecA, femA, and femB genes in clinical strains of staphylococci using polymerase chain reaction.

MecA, a structural gene located on the chromosome of Staphylococcus aureus, characterizes methicillin-resistant S. aureus (MRSA), and femA and femB(fem) genes encode proteins which influence the level of methicillin resistance of S. aureus. In order to examine effectiveness of detecting mecA and fem genes in identification of MRSA, the presence of these genes in 237 clinically isolated strains of staphylococci was investigated by polymerase chain reaction (PCR). An amplified mecA DNA fragment of 533 base pairs (bp) was detected in 100% of oxacillin-resistant S. aureus, in 16.7% of oxacillin-sensitive S. aureus, in 81.5% of S. epidermidis, and in 58.3% of other coagulase-negative staphylococci (CNS). While the PCR product of femA (509 bp) or femB (651 bp) was obtained from almost all the S. aureus strains except for five oxacillin-resistant strains (2.5%), neither of these genes were detected in CNS. Therefore, the detection of femA and femB together with mecA by PCR was considered to be a more reliable indicator to identify MRSA by differentiating it from mecA-positive CNS than single detection of mecA.

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.

New P serotype of group A human rotavirus closely related to that of a porcine rotavirus

The VP7 and VP4 genes of two human group A rotavirus strains Mc323 and Mc345 with unique serologic and genomic properties, and isolated in Chiang Mai, Thailand, in 1989 [Urasawa et al. (1992) Journal of Infectious Diseases 166:227–234] were further characterized. The nucleotide and deduced amino acid sequences of the VP7 genes allowed the classification of both strains as serotype G9. The VP4 genes of both strains are 2,359 nucleotides in length and encode a protein of 775 amino acids like in most human rotaviruses. A comparison of the VP4 amino acid sequence of strain Mc323 with those of strain Mc345 and 24 human and animal rotaviruses representing 20 distinct VP4 genotypes reported to date showed that VP4 of Mc323 and Mc345 belong to genotype 19 previously reported for porcine rotavirus [Burke et al. (1994) Journal of General Virology 75:2205–2212]. To investigate the serological type (P serotype) of these VP4s, six reassortant viruses each containing a distinct VP4 gene characteristic of human rotaviruses and the VP7 gene of porcine rotavirus strain Gottfried (G4) were prepared, and antisera to these reassortants produced in rabbits. In neutralization tests, the P serotype of Mc323 was clearly differentiated from the five major P serotypes reported previously for human rotaviruses, suggesting that Mc323 and Mc345 represent a new human rotavirus P serotype tentatively called P11. J. Med. Virol. 60:63–69, 2000.

OUTBREAK OF INFANTILE GASTROENTERITIS DUE TO TYPE 40 ADENOVIRUS

Genetic and antigenic characterisation was performed on a strain of adenovirus (EAd) isolated from an outbreak of gastroenteritis which occurred in an orphanage in the City of Sapporo, in the room housing the eldest children, who ranged in age from 14 to 22 months. 7 of the 11 children housed in that room had diarrhoea between July 11 and July 22, 1982. All 7 shed adenoviruses detectable by electron microscopy in their stools. Immune electron microscopy showed that all patients as well as the healthy contacts sharing the room underwent seroconversion to EAd. There was no homology, or very slight homology, between DNA of EAd and those of adenoviruses belonging to subgroups A to E. Antigenically EAd was closely related to type 40 adenovirus, so far the sole member of the newly identified subgroup F. This outbreak of gastroenteritis is the first in which the causative agent has been identified as being a member of subgroup F adenoviruses.

Survey on the distribution of the gene 4 alleles of human rotaviruses by polymerase chain reaction

The presence of six gene 4 alleles (or VP4 genotypes) in human rotaviruses has been recognized. Using 16 representative cultivable human rotavirus strains, we confirmed the specificity of VP4 genotyping by polymerase chain reaction (PCR) using the nested oligonucleotides specific to each of the four representative gene 4 alleles. Using the PCR, we surveyed the gene 4 alleles of 199 human rotaviruses in stools collected in Japan and Thailand. Strains with the gene 4 allele, corresponding to P1A serotype, were shown to be the most prevalent, but two strains with P2 gene 4 allele and one strain with P3 gene 4 allele were detected in Thailand and in Japan, respectively.

Serotype determination of human rotavirus isolates and antibody prevalence in pediatric population in Hokkaido, Japan

SummaryThree different serotypes of human rotavirus isolates defined in our laboratory were compared by cross neutralization tests with human rotavirus serotypes established in the NIH, U.S.A. The results clearly demonstrated that our three serotypes correspond well to their serotypes Wa, DS-1 and M (or P). Using the three serotype-specific rabbit antisera, all of the 16 strains isolated to date could be assigned to one of those three serotypes.The prevalence of human rotavirus serotypes 1, 2 and 3 among inhabitants of Sapporo and its outskirts was investigated based on the results of neutralizing antibody distribution patterns by age using sera of non-infectious disease patients examined at the Sapporo Medical College Hospital. Neutralizing antibody titers were measured against four strains, KU and K8 (serotype 1), S2 (serotype 2) and YO (serotype 3).The results revealed that serum antibody titers against KU, K8 and YO strains rose with time after birth, reaching the highest antibody distribution levels in either the 3–5-year-old or 6–9-year-old age group, while antibody against S2 strain tended to be lower than that against the other three strains throughout all age ranges examined, with the highest level being shown in the adult group.

Serological and genomic characterization of human rotaviruses detected in China

A total of 1,385 stool specimens were collected from children with diarrhea at two hospitals in Wuhan, Hubei Province, China, in 1994 and 1995, and screened for rotavirus by polyacrylamide gel electrophoresis of viral RNA. Group A rotavirus was detected with high frequency; 56.5% (87/154) and 40.8% (502/1,231) of the specimens collected in 1994 and 1995, respectively, were positive for rotavirus. Assignment of G serotype and P type (VP4 genotype) of group A rotavirus by ELISA with monoclonal antibodies and/or PCR, respectively, showed that strains of G2‐P[4] and G1‐P[8] specificity were predominant in 1994 and in 1995, respectively. In contrast, a single strain was found to have a P[9] type specificity, and no G4 strain was detected. Unusual combinations of RNA pattern‐subgroup‐G serotype‐P type, such as long pattern‐subgroup I‐G1‐P[8], short pattern‐subgroup II‐G3‐P[4] and short pattern‐subgroup I‐G1‐P[4], were detected in four specimens. Nucleotide sequences of the VP8* and/or NSP5 genes from two Chinese P[8] strains 470 and 582 and one Chinese P[9] strain 512 as well as five Japanese P[9] strains (K8, AU1, M318, O264, and O265) were determined and compared with the published sequences of the corresponding gene. In the phylogenetic tree of VP8* sequences of P[9] strains, which formed two clusters each having strain K8 or AU‐1 as the representative strain, the Chinese P[9] strain was found in the cluster represented by AU‐1, although it was most distantly related to other strains. While NSP5 sequences of human strains with P[9] specificity were related to simian and bovine strains, that of Chinese P[8] strains was most closely related to those of porcine strains. A single group C rotavirus (No. 208) was detected. Nucleotide sequences of its VP4, VP6, VP7, and NSP4 genes were very similar to those of group C human rotaviruses detected worldwide. J. Med. Virol. 55:168–176, 1998.

The distribution of G and P types within isolates of bovine rotavirus in Japan.

Various combinations of G type and P type were observed in 76 bovine rotavirus (BRV) strains isolated from 235 diarrheal calves in three prefectures of Japan in 1992-1994. The most prevalent combination was G6:P5 (46/76, 60.5%), followed by G10:P11 (13/76, 17.1%), G6:P11 (7/76, 9.2%) and G10:P5 (5/76, 6.6%). No G6:P1 strain of BRV was recognized from the isolates in the present study, though this type of BRV is well known as a suitable vaccine strain against BRV infection.