Koki Taniguchi

Evolutionary history and global spread of the emerging g12 human rotaviruses.

ABSTRACT G12 rotaviruses were first detected in diarrheic children in the Philippines in 1987, but no further cases were reported until 1998. However, G12 rotaviruses have been detected all over the world in recent years. Here, we report the worldwide variations of G12 rotaviruses to investigate the evolutionary mechanisms by which they managed to spread globally in a short period of time. We sequenced the complete genomes (11 segments) of nine G12 rotaviruses isolated in Bangladesh, Belgium, Thailand, and the Philippines and compared them with the genomes of other rotavirus strains. Our genetic analyses revealed that after introduction of the VP7 gene of the rare G12 genotype into more common local strains through reassortment, a vast genetic diversity was generated and several new variants with distinct gene constellations emerged. These reassortment events most likely took place in Southeast Asian countries and spread to other parts of the world. The acquirement of gene segments from human-adapted rotaviruses might allow G12 to better propagate in humans and hence to develop into an important emerging human pathogen.

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.

Rotavirus VP7 neutralization epitopes of serotype 3 strains

Sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) was performed on 27 human and animal rotavirus strains of serotype 3 in order to examine genetic variation within strains of identical serotype. Comparisons of the deduced amino acid sequences of the VP7s showed overall sequence identities of 85% or higher. A higher degree of overall VP7 sequence similarity was observed among strains from the same animal species when compared to strains from different animal species, suggesting that there are species-specific sequences in the VP7 protein. Alignment of the amino acid sequences demonstrated that amino acid sequence divergence among serotype 3 strains from different species was located primarily in previously established VP7 serotype-specific regions where genetic variation was identified among strains of different serotype. These regions were highly conserved among serotype 3 strains derived from the same species. The varying reactivities of three anti-VP7 monoclonal antibodies with the 27 strains was consistent with the occurrence of antigenic variation among serotype 3 strains. Moreover the reactivity of monoclonal antibodies correlated with the amino acid sequence found in two serotype-specific regions (VR5 and VR8). A computer-derived predicted phylogenetic tree suggests that rotavirus strains from different animal species belonging to serotype 3 are more closely related to each other than to rotavirus strains of different serotypes.

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.

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.

Unusual human rotavirus strains having subgroup I specificity and “long” RNA electropherotype

SummaryDuring an epidemiological study of human rotavirus in Metro Manila, Philippines, we found 20 unusual strains which belong to subgroup I but have “long” RNA pattern typical of subgroup II human rotavirus. The RNA patterns of the 20 strains were classified into four groups though they were very similar to each other. Four strains, designated L4, L26, L27, and L34 were isolated in MA 104 cells from stool specimens. They possessed subgroup I specificity and long RNA pattern identical to that of the viruses in the original stool samples. The serotype specificity of these strains could not be determined by either enzyme-linked immunosorbent assay (ELISA) or neutralization test, while one strain (L27) was neutralized by serotype 2-specific anti-VP4 monoclonal antibody. These strains were suggested to have an unusual antigenicity on VP7.

Analysis of the newly identified neutralization epitopes on VP7 of human rotavirus serotype 1

Neutralizing monoclonal antibodies (MAbs) directed to the VP7 protein and neutralization-resistant mutants were used to analyse the antigenic structure of VP7 of human rotavirus serotype 1. Cross-neutralization tests using the MAbs and the resistant mutants indicated the existence of two functionally independent neutralization epitope regions (S1 and S2) on VP7. Region S1 corresponds to a single epitope domain of VP7 which has been detected previously. Two MAbs prepared in this study recognized the S1 region, and the resistant mutants they selected had amino acid substitutions at positions 94 or 213. On the other hand, region S2 is considered to be a novel epitope. Single or double amino acid substitutions were detected in the variable regions (amino acid positions 145, 217 and 221) and in the constant regions (positions 104, 201 and 291) of the VP7 protein of mutants selected by MAbs directed to the S2 region. It was suggested that the variable region E (amino acids 208 to 221) includes two independent neutralization sites, and that amino acid substitutions in the constant region of VP7 also affect serotype-specific neutralization epitopes. Neutralization epitopes on VP7 are considered to be highly dependent on the conformation of VP7.

Identification of operationally overlapping and independent cross-reactive neutralization regions on human rotavirus VP4

Cross-reactive neutralization epitopes on VP4 of human rotavirus (HRV) were analyzed by the use of VP4-specific neutralizing monoclonal antibodies (N-MAbs) and MAb-resistant mutants. Seven anti-VP4 N-MAbs obtained in this study by using HRV serotypes 1 and 3 as immunizing antigens showed a variety of cross-reactivity patterns to 20 HRV strains with different serotype specificity in neutralization tests and a broader cross-reactivity to them was found for four N-MAbs in an enzyme-linked immunosorbent assay. On the basis of the reactivity patterns against rotaviruses in neutralization tests, these seven N-MAbs were classified into four groups. Cross-neutralization tests using a total of 12 pairs of MAbs and resistant mutants, including five pairs which had been prepared previously, showed that VP4 of HRV (strain KU) contained two independent antigenic regions. One, region C1, was recognized by a single MAb (YO-2C2) and the other was made up of two antigenic regions (C2 and C3) which overlapped operationally. Identification of amino acid substitution sites on VP4 of representative mutants of HRV strain KU indicated that amino acid positions 385 or 392 and 428 or 433 were critical for the C2 and C3 regions, respectively. These results suggested that regions C2 and C3 exist as conformational antigenic sites.