Tomoko 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.

Identification of human and bovine rotavirus serotypes by polymerase chain reaction.

The use of the polymerase chain reaction (PCR) for identifying serotypes of human and bovine rotaviruses was examined. In the identification of 115 human rotavirus samples in stools, results with PCR showed excellent agreement with results of serotyping by an enzyme-linked immunosorbent assay (ELISA) using serotype-specific monoclonal antibodies. Furthermore, the PCR showed a much higher sensitivity (93%) than the ELISA test (82.6%). The PCR method could also be applied for identifying the serotype of bovine rotaviruses.

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.

Molecular and Antigenic Analyses of Serotypes 8 and 10 of Bovine Rotaviruses in Thailand

Antigenic and genomic properties of non-serotype 6 bovine rotaviruses isolated in Thailand and Japan were studied by cross-neutralization tests, nucleotide sequence determination of the VP7 gene, and RNA-RNA hybridization. Two Thai strains (61A and A44) were serologically related to a Japanese isolate KK3 which has been assigned to serotype 10. In contrast, strain A5 was found to be antigenically similar to human strain 69M with serotype 8 specificity, although strain A5 showed a one-way cross-reaction with serotype 6 strain NCDV. VP7 sequence analysis confirmed these results. High degrees of similarity in nucleotide and amino acid sequences (92.5 to 98.2% and 96.3 to 97.9%, respectively) were found among the VP7 genes of the four serotype 10 bovine strains (61A, A44, KK3 and B223). The VP7 amino acid sequence of strain A5 was similar to those of serotype 8 human strains (91.7% and 94.8% for strains B37 and 69M, respectively). In RNA-RNA hybridization experiments, a high level of overall relatedness was found among the three serotype 10 bovine strains (61A, A44 and KK3), and strains A5 and NCDV were also moderately related to the three serotype 10 viruses. All the bovine rotaviruses tested in this study, regardless of their serotype specificity, exhibited a moderate genetic-relatedness to strain 69M of serotype 8, and, to a lesser extent, to serotype 2 human rotavirus strains.

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.

Presumptive seventh serotype of human rotavirus

SummaryFour group A human rotaviruses having antigenic specificity of subgroup I and ‘long’ RNA electropherotype were isolated in MA104 cell cultures. Cross-neutralization tests with hyperimmune antisera suggested that they are serologically distinct from the six previously recognized human rotavirus serotypes.

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.