Kenichi Umene

Quantitative analysis of genomic polymorphism of herpes simplex virus type 1 strains from six countries : studies of molecular evolution and molecular epidemiology of the virus

Using the presence or absence of 63 variable restriction endonuclease (RE) sites selected from 225 sites with six REs, genomic polymorphism of 242 herpes simplex virus type 1 (HSV-1) strains from six countries (Japan, Korea, China, Sweden, U.S.A. and Kenya) was quantitatively analysed. Twenty-five of the 63 sites were found to differ between Korean and Kenyan strains. In contrast, only three and six sites were found to differ between isolates from Sweden and the U.S.A. and between those from Korea and China, respectively, suggesting that they are closely related to each other. In this way, characterization of 63 sites enabled us to categorize 186 distinct HSV-1 genotypes from 242 individuals. Some strains from Japan, Korea and China shared the same genotypes, indicating that they are phylogenetically closely related. Many significant correlation coefficients (magnitude of > 0.42; P < 0.01) between pairs of sites were found in isolates from the three Asian countries (Japan, Korea and China) as well as in those from Sweden and the U.S.A., suggesting that HSV-1 strains from within the same ethnic groups are evolutionarily closer. The average number of nucleotide substitutions per nucleotide, as defined by nucleotide diversity (pi), was estimated for HSV-1 genomes within (pi x or pi y) and between (pi xy) countries. On the basis of 225 sites, nucleotide diversity for Kenyan isolates was 0.0056, almost three times higher than that for Korean isolates, implying that Kenyan HSV-1 genomes are much more diverse than those from Korea. In addition, the diversity between HSV-1 isolates from different countries (pi xy) was highest between isolates from the three Asian countries and Kenya (0.0075 to 0.0081) and lowest among those from the three Asian countries (0.0032 to 0.0040). The mutation rate (lambda) for HSV-1 was estimated to be 3.5 x 10(-8)/site/year. All these findings show that the evolution of HSV-1 may be host-dependent and very slow.

Mechanism and application of genetic recombination in herpesviruses

Herpes simplex virus type 1 (HSV‐1) is a ubiquitous human pathogen that latently infects sensory ganglia and encodes over 80 genes in a 152 kbp DNA genome. This well characterised virus provides a model for analysing genetic recombination in herpesviruses, a fundamental biological process by which new combinations of genetic materials are generated. The frequency of homologous recombination was estimated to be 0·0048–0·007 (0·48%–0·7%)/kb of the HSV‐1 genome, determined using physical markers. The double‐strand break repair model, the current model of homologous recombination, adequately explains L‐S inversion of herpesvirus genomes and the recombinogenicity of the a sequence. Several herpesvirus genomes, including HSV‐1 consist of a unique sequence bracketed by a pair of inverted repeat sequences. This arrangement is attributed to illegitimate recombination between molecules arranged in an inverse orientation. Junctions of unique and repeated sequences that correspond to the crossover site of illegitimate recombination are recombinogenic. Recombination is important for virus evolution, construction of mutated virus, gene therapy and vaccination in which the potential for recombination between engineered input virus and wild type virus has to be considered. Copyright

Evolution of herpes simplex virus type 1 under herpesviral evolutionary processes

SummaryHerpesviruses, the genomes of which are double-stranded DNA of 120 kilobase pairs or more, infect a wide range of vertebrates from mammals to fish. Herpes simplex virus type 1 (HSV-1), a representative of family Herpesviridae, is a ubiquitous human pathogen. HSV-1 relates to common muco-cutaneous diseases, while HSV-1 infection can mean a serious outcome, e.g. blindness and insult to the central nervous system. Evolution of herpesviruses includes DNA rearrangements, often generating tandemly or invertedly repeated sequences. Studies of HSV-1 DNA dynamics substantiated these processes of DNA recombination involved in the evolution of herpesvirus. Herpesviruses seem to have diversified from a common ancestor, in a manner mediating co-speciation of herpesviruses with host species through species-specific latent infections. Thus, the notion of host-linked evolution of herpesviruses is given support. Relationships between HSV-1 genotypes and human ethnic groups can be traced by analyses of DNA polymorphisms of HSV-1 strains present in populations of various countries. A close association of an HSV-1 genotype with a particular historical human population seems probable. Such being the case, the host-linked mode is likely to be linked to diversification of HSV-1 in human populations.

A new genome type of human parvovirus B19 present in sera of patients with encephalopathy.

The possible involvement of human parvovirus B19 infection in encephalopathy has been reported. To determine the characteristics of B19 viruses involved in such cases, we molecularly cloned a part of the B19 DNAs derived from the sera of three patients with encephalopathy (B19 strains N80, N81 and N82), following amplification by PCR. The nucleotide (nt) sequences of the cloned DNAs (nt 3147-3405) were then determined. The nucleotide sequence of N80 was similar to that of the known genome type of group II. The nucleotide sequences of N81 and N82 were similar to each other, but distinctly different from those of other B19 strains reported previously. Almost the entire genome of strain N81 (nt 229-4812) was molecularly cloned following amplification by PCR. Analyses of the restriction site polymorphisms of the cloned N81 DNAs showed that N81 is distantly related to other B19 strains. Therefore, the two strains of N81 and N82 were defined as belonging to a new genome type, group V. Group V B19 virus has so far been isolated only from patients with encephalopathy.

The genome type of human parvovirus B19 strains isolated in Japan during 1981 differs from types detected in 1986 to 1987: a correlation between genome type and prevalence

The genome DNAs of 12 strains of human parvovirus B19, isolated in Japan at two different times, 1981 and 1986 to 1987, were molecularly cloned in the plasmid pUC18. The cloned B19 DNAs were analysed by cleaving with restriction endonucleases, and were classified into several groups by genome type. The restriction endonuclease cutting patterns of B19 strains isolated during 1981 were similar to that of the group IV genome type, and the patterns of those isolated later were similar to that of group II, suggesting a correlation between the genome type and the prevalence. We conclude that the prevalences of B19 infection in Japan during 1981 and in 1986 to 1987 were caused by viruses differing in genome type, and that B19 viruses with similar genome types disseminated widely in Japan during each prevalence.

Intermolecular Recombination of the Herpes Simplex Virus Type 1 Genome Analysed Using Two Strains Differing in Restriction Enzyme Cleavage Sites

Intermolecular recombination of herpes simplex virus type 1 (HSV-1) was studied by analysing the segregation of strain-specific restriction enzyme cleavage sites among progeny viruses produced after co-infection by two HSV-1 strains differing in eight restriction enzyme cleavage sites. Out of 93 progeny viruses examined, 51 clones were recombinant, and crossover sites of the recombinants were mapped on the HSV-1 genome. These sites were distributed evenly in the unique sequence of the L component (UL) and the recombination frequency in UL was estimated to be 1.12 per genome length, or 0.007 per kilobase pair. No evidence was obtained to support the existence of enhanced intermolecular recombination events in the regions containing inverted repeats and the L-S junction in comparison with the recombination frequency in UL. The finding of recombinants in an arrangement that minimized the number of crossover events suggested the participation of both of two arrangements of the L component of parental DNA (P or IS, and IL or ISL) in the generation of the recombinants. The possibility of a preference for P or IS over IL or ISL arrangements remains to be determined.

Herpes simplex virus type 1 restriction fragment polymorphism determined using southern hybridization

SummaryRegions of herpes simplex virus type 1 (HSV-1) DNA with variation in the size of restriction endonuclease fragments were identified by comparison of theBam HI,KpnI orSalI restriction endonuclease digestion patterns among 15 HSV-1 isolates after hybridization with specific32P-labeled cloned HSV-1 DNA fragments. Of the types of restriction fragment polymorphism identified, one was a strain with a distinctly different restriction fragment than the prototype (loss or gain of restriction sites). Another type, the specific fragment varied only in size among strains. Thirteen distinct variations were identified. Ten were mapped to the unique sequence of the L component; two to the inverted repeat of the L component and one to the inverted repeat of the S component. The presence of a common ancestor from which some isolates of HSV-1 might derive was deduced from an analysis of the distribution of the thirteen variations among the 15 HSV-1 isolates.

Genomic characterization of two predominant genotypes of herpes simplex virus type 1.

SummaryGenomic profiles of 66 strains of herpes simplex virus type 1 (HSV-1) isolated in Japan were investigated with regard to restriction fragment length polymorphism (RFLP) and length variation of fragments containing reiterations. There were two predominant genotypes of F1 and F35, and the genomic characteristics of each were studied. The nucleotide change between F1 and F35 was estimated to be 1.5%. An RFLP marker (VR23) peculiar to genotype F35 was identified as the first case of genomic marker specific to a predominant genotype of HSV-1, and is the diagnostic marker of F35. Thea sequences (repeating in an HSV-1 genome and containing reiterations) of F35 were cleaved by Sac II on the DR4 (direct repeat 4) stretch, whilea sequences of F1 had a rearranged DR4 and were resistant to Sac II digestion. Thus, analyses of fragments containing reiterations, such asa sequences, can serve to classify HSV-1 strains as well as for purpose of differentiation. The proportion of strains derived from primary infection to those from recurrent infection was higher in strains of F35 than in those of F1, and this genotypic difference within HSV-1 may possibly influence clinical manifestations.

Genetic Diversity of Human Parvovirus B19 Determined Using a Set of Restriction Endonucleases Recognizing Four or Five Base Pairs and Partial Nucleotide Sequencing: Use of Sequence Variability in Virus Classification

Analysis of the restriction site polymorphism (RSP) of human parvovirus B19 using 12 restriction endonucleases (REs) recognizing four or five bp sequences (4- or 5-bp REs) revealed a significant difference between strains previously classified as being of the same genome type, and a relationship between two strains of different genome types, thereby indicating a global spread of B19 virus strains. These findings demonstrate the advantage of this set of 4- and 5-bp REs for the calculation of the degree of genetic diversity and clearly it is necessary to amend the taxonomy of B19 virus strains using these REs. We examined the nucleotide (nt) sequence between nt 3141 and 3411, at the N terminus of the VP2 protein coding region, in 12 B19 virus strains. The pattern of distribution of nucleotide differences between the strains confirmed the classification by RSP analysis. Between nt 3293 and nt 3364, a region in which an antigenic epitope may be encoded, there was no evidence of a nucleotide change causing an amino acid change. Thus, the amino acid sequence in this potential epitope is probably conserved.

Populations of two Eastern countries of Japan and Korea and with a related history share a predominant genotype of herpes simplex virus type 1

SummaryHerpes simplex virus type 1 (HSV-1), a common human pathogen of non-epidemic nature is linked closely to the individual by latent infection. HSV-1 genotypes usually differ with race. Based on a “dual structure model” for population history of the Japanese, modern Japanese populations are assumed to have derived from two major migration events. The Jomon people arrived in Japan >10,000 years ago and the Yayoi people began migrating to Japan from the Korean peninsula ∼2,300 years ago. The presence of two predominant genotypes of F1 and F35 was noted in HSV-1 strains isolated in Japan. As the F1 genotype also predominated in Korea, peoples of Japan and Korea share the F1 genotype. Regional differences in the frequency of F1 and F35 genotypes within Japan seem to relate to differences in the dispersion of descendants of the Yayoi and Jomon peoples. Our hypothesis is that the F35 genotype relates to the Jomon people, earlier residents in Japan, while the F1 genotype relates to the Yayoi people who migrated much later from the Korean peninsula.