Osamu Nakagomi

Identification of rotavirus genogroups by RNA-RNA hybridization

The genetic relatedness of various human rotavirus strains was examined by RNA-RNA hybridization in which 32P-labelled single stranded RNAs produced by in vitro transcription from viral RNAs were used as probes. Denatured genomic double stranded RNAs were hybridized to the probes under highly stringent conditions and the resulting hybrids were fractionated by polyacrylamide gel electrophoresis. Based on the hybridization patterns obtained with probes made from prototype strains Wa (subgroup II, long RNA electropherotype), DS-1 (subgroup I, short RNA electropherotype) and AU-1 (subgroup I, long RNA electropherotype), we have observed that human rotaviruses fall into three distinct gene groups which we have termed genogroups. Identification of genogroups among rotavirus isolates will prove to be a valuable asset for the analysis of naturally occurring reassortants, to trace interspecies transmission of animal rotaviruses to man or vice versa and to identify rotaviruses from environmental sources with regard to their original host species. Furthermore, such an approach will contribute to our understanding of the evolution of rotavirus genes.

Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): Application to sequencing 6·5 kb genome segment of hantavirus strain B-1

A method, referred to as cassette-ligation mediated polymerase chain reaction (PCR), has been developed to permit selective and specific amplification of cDNA sequence from total cellular RNA. This technique comprises (i) digestion of cDNA with multiple restriction enzymes, (ii) ligation of cleavage products to double-stranded DNA cassettes possessing a corresponding restriction site and (iii) amplification of cassette-ligated restriction fragments containing a short, known sequence (but not all the other ligation products) by PCR using the specific and cassette primers; the specific primer is designed to prime synthesis from the known sequence of the cDNA whereas the cassette primer anneals to one strand of the cassette. Sequencing from the cassette primer provides information to design a new primer for the next walking step. The amplified cDNA fragments are often larger than the maximum DNA fragments (500-600 bp) that can be sequenced without the need of synthesizing internal sequencing primer. Each of such large cDNA fragments is dissected into smaller DNA fragments by repeating cassette-ligation mediated PCR exploiting different restriction sites and different sets of cassette primers. This dissection process reduces the number of specific primers to a minimum, thereby increasing the speed of sequencing and minimizing the overall cost. We have successfully applied this cDNA walking and sequencing by the cassette-ligation mediated PCR to the sequencing of an entire 6.5 kb genome segment of hantavirus strain B-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of anti-rotavirus activity of extract from Stevia rebaudiana

Anti-human rotavirus (HRV) activity of hot water extracts from Stevia rebaudiana (SE) was examined. SE inhibited the replication of all four serotypes of HRV in vitro. This inhibitory effect of SE was not reduced on the prior exposure of SE to HCl for 30 min at pH 2. Binding assay with radiolabeled purified viruses indicated that the inhibitory mechanism of SE is the blockade of virus binding. The SE inhibited the binding of anti-VP7 monoclonal antibody to HRV-infected MA104 cells. The inhibitory components of SE were found to be heterogeneous anionic polysaccharides with different ion charges. The component analyses suggested that the purified fraction named as Stevian with the highest inhibitory activity consists of the anionic polysaccharide with molecular weight of 9800, and contains Ser and Ala as amino acids. Analyses of sugar residues suggest uronic acid(s) as sugar components. It did not contain amino and neutral sugars and sulfate residues. These findings suggest that SE may bind to 37 kD VP7 and interfere with the binding of VP7 to the cellular receptors by steric hindrance, which results in the blockade of the virus attachment to cells.

Incidence and Burden of Rotavirus Gastroenteritis in Japan, as Estimated from a Prospective Sentinel Hospital Study

We assessed the burden of rotavirus infection-related disease, in terms of hospitalization and associated costs, at 3 sentinel hospitals in Akita prefecture, Japan. From January 2001 through December 2002, a total of 443 children <5 years of age were hospitalized for acute gastroenteritis. Of 422 stool specimens collected, 244 (58%) tested positive for rotavirus. Only 7.8% of the rotavirus disease-associated hospitalizations involved infants <6 months of age, whereas most cases of disease (39%) were reported in the second year of life, and 89% of cases had occurred by 36 months of age. The mean severity score for rotavirus gastroenteritis resulting in hospitalization was 16.5, according to the modified 20-point severity scoring system. The average associated direct medical cost was 136,000 yen (1236 US dollars) per case and was similar among the 3 hospitals. The estimated incidence of rotavirus disease-associated hospitalizations among children <5 years of age was 7.9-17.6 hospitalizations/1000 person-years, and the estimated cumulative incidence by 5 years of age was 6.6%. Thus, approximately 1 in 15 children will require hospitalization due to rotavirus diarrhea by their fifth year of life. In Japan, this would mean that 78,000 children <5 years of age would be hospitalized each year, resulting in a direct medical cost of 10 billion yen (96 US dollars million). The burden associated with rotavirus gastroenteritis in Japan is substantial and might be reduced through the introduction of vaccines.

Intragenic recombinations in rotaviruses

In this paper, evidence for intragenic recombination in the VP7 gene between rotavirus strains bearing different serotypes is demonstrated for the first time. Intragenic recombination may be one of the escaping mechanisms from the host immune system for rotavirus. This process involves exchanging antigenic regions, thus questioning the use of multivalent vaccines for the prevention of rotavirus infection.

Interspecies Transmission of Rotaviruses Studied from the Perspective of Genogroup

Group A rotaviruses, members of the genus Rotavirus in the family Reoviridae, have been implicated as the single most important etiological agent of severe diarrhea in infants and young children worldwide (32). Rotaviruses were known to have a wide host range as indicated by their recovery from almost every animal species when appropriate techniques for virus detection were employed (32). When certain animal rotavirus strains, such as feline and canine strains, share serotype G3 with some human strains, speculation on the role of animals as a source of rotavirus infections of humans has intensified (32). Further studies revealed that serotype G3 rotaviruses have the broadest host range which includes humans, monkeys, dogs, cats, horses, pigs, mice, and rabbits. This speculation was weakened, however, when Flores et al (12, 14) demonstrated by RNARNA hybridization assays that most of the corresponding genes of animal and human rotaviruses do not share a high degree of homology that is observed among rotaviruses recovered from the same animal species. Furthermore, sequence analysis of the gene encoding the major neutralization glycoprotein (VP7) performed on 27 human and animal rotavirus strains of serotype G3 revealed that a higher degree of homology is observed among strains from the same animal species when compared to strains from different animal species (58). These observations have formed the basis for the prevailing view, such as the one stated in the rotavirus chapter of Fields Virology (32), that animal rotaviruses do not infect humans or human rotaviruses do not infect animals under natural conditions. As a logical consequence to the concept that rotaviruses have a restricted host range in nature, animal rotaviruses are considered to have lower fitness in human tissues in terms of the efficiency of replication and, therefore, they are believed to be naturally attenuated for humans. This led to the consideration of the Jennerian approach to vaccination in which an animal strain is used to immunize humans against diarrheal disease caused by human rotaviruses (31). Ever since we encountered an unusual human rotavirus strain which was isolated from an infant with diarrhea in Akita, Japan, in 1982 (43), we have held the contrary view that animal rotaviruses can infect humans and cause disease whenever the chance exists. This strain which we named AU1 (named after Akita University) had a combination of serotype G3, subgroup I and a long RN A pattern (33, 44). Until the discovery of the AU-1 strain, such combination had never been observed in human rotaviruses but was quite typical of animal rotavirus strains (20). After various possibilities of laboratory contamination were carefully excluded (44), the AU-1 strain, together with the AU228 strain which was isolated from a child with a clinical history of contact with a cat, was shown by RNA-RNA hybridization to be more closely related to a feline rotavirus strain (strain FRV-1) isolated in Kagoshima, Japan, than to any other human rotaviruses (56). Much evidence is now accumulating which suggests that interspecies * Address correspondence to Dr . Osamu Nakagomi, Department of Microbiology, Akita University School of

Genetic diversity and similarity among mammalian rotaviruses in relation to interspecies transmission of rotavirus

SummaryTo address the question whether there was any molecular evidence for interspecies transmission of rotaviruses from one animal species to another, genetic relationships among human and animal rotaviruses were examined by a series of hybridization experiments in which genomic RNAs from 14 rotavirus strains derived from seven different host species were hybridized with the [32P]-labelled transcription probes prepared from 11 strains representing rotaviruses from those seven host species. In general, higher level of homology among most, if not all, of the cognate gene segments that allowed classification into the same genogroup was shared among rotaviruses recovered from the same animal species but this level of homology was not found among rotavirus strains derived from different host species. However, such a high level of homology that was usually found among rotaviruses recovered from the same animal species was detected between feline rotavirus strain Cat97 and canine rotavirus strain K9 as well as between human rotavirus strain AU-1 and feline rotavirus strain FRV-1. The sharing of closely related genetic constellation of most of the 11 gene segments (genogroup) by rotaviruses recovered from different animal species provided molecular evidence that interspecies transmission of rotaviruses occurred in nature at least recently in the evolutionary history.

The Relative Frequencies of G Serotypes of Rotaviruses Recovered from Hospitalized Children with Diarrhea: A 10‐Year Survey (1987–1996) in Japan with a Review of Globally Collected Data

Since rotavirus vaccines aim to protect children from severe diarrhea, knowledge of the prevailing G serotypes among rotaviruses from hospitalized children is essential. Thus, we determined the G serotypes of rotaviruses collected from children with acute diarrhea in a local referral hospital in Akita, Japan, over the 10‐year period between January 1987 and December 1996. Based on the assumption that rotaviruses with an identical electropherotype possess the same G serotype, the G serotypes of 488 rotavirus‐positive specimens that were classified into 63 electropherotypes were determined by enzyme‐linked immunosorbent assay with a supplementary use of G typing by reverse transcription‐PCR. The relative frequencies over the 10‐year period were 77.0 (G1), 14.5 (G2), 2.7 (G3) and 5.3% (G4), leaving the possibility that only 0.4% had G serotypes uncommon to human rotaviruses. Of 24,050 rotaviruses extracted by reviewing 63 serotyping studies in literature, the relative frequencies of the four major G serotypes were 50.6 (G1), 9.3 (G2), 7.2 (G3) and 11.6% (G4). As to uncommon G serotypes, only 0.9% were described as serotypes other than G1–4, and our estimate for potential uncommon serotypes were at most 8.1%. Thus, both this long‐term study focusing on the rotaviruses only from severe cases in a single hospital in Japan and the global review of G serotypes published to date indicate that the primary target of any rotavirus vaccines should be rotaviruses possessing serotypes G1–4.

Characterization of human rotavirus genotype P(8)G5 from Brazil by probe-hybridization and sequence

SummaryWe report the molecular characterization of rotavirus genotype P[8]G5 strains found in fecal specimens collected in four different regions of Brazil, using digoxigenin (dig)-labeled oligonucleotide probes, sequence analysis, and RNA-RNA hybridization. The closest sequence relationships of the neutralization antigens of these strains were to the VP4 protein of P1A[8]G1 strain KU (93.3% identity in amino acids 11 to 282) and to the VP7 protein of G serotype 5 strain OSU (87.6% identity in amino acids 8 to 232). Based on VP7 sequence differences, we designed dig-probes that allowed us to discriminate porcine OSU-like strains from G5 strains isolated from Brazilian infants. The genetic relationships of two P[8]G5 isolates to other rotavirus genogroups were analyzed by RNA-RNA hybridization with [32P]-GTP probes representative of serotypes P1A[8]G1 (Wa), P[8]G3 (AU17), and P9[7]G5 (OSU). The Brazilian P[8]G5 strains showed sequence homology with genes of Wa-like and OSU-like strains, suggesting that these two strains were naturally occurring reassortants between members of the Wa and porcine rotavirus genogroups. The identification of these strains in diverse geographic areas of Brazil underscores their stability and demonstrates the emergence of clinically important rotavirus diarrhea strains by reassortment.

Human and bovine serotype G 8 rotaviruses may be derived by reassortment

SummaryThe origin of, and relationship between human and bovine serotype G 8 rotaviruses were investigated by genomic hybridisation. Radiolabelled mRNAs of human G 8 rotaviruses 69 M (isolated in Indonesia) and HAL 1271 (isolated in Finland), and bovine rotaviruses KK 3 (G 10) and NCDV (G 6), were used as probes. The products of liquid hybridisation between the probes and the genomic RNA of human and bovine rotaviruses, including bovine G 8 rotavirus 678 (isolated in Scotland) and two other Finnish human G 8 rotaviruses HAL 1166 and HAL 8590, were examined by separation in polyacrylamide gels. The genomes of Finnish human G 8 rotaviruses were similar to those of bovine G 6 and G 10 rotaviruses. Neither Indonesian human G 8 nor bovine G 8 viruses had high levels of similarity to each other or to other bovine and human rotaviruses. Thus these three epidemiologically distinct G 8 rotaviruses have different origins and may be derived by reassortment with rotaviruses of a third, as yet unknown, host species. The similarity between the Finnish isolates and the bovine isolate NCDV suggests that they have diverged recently and that these human G 8 rotaviruses may be derived from a zoonotic infection, or alternatively, from the live rotavirus vaccine of bovine origin which has been used to vaccinate Finnish children.