Hiroshi Sakaoka

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.

Disseminated infection of herpes simplex virus with fulminant hepatitis in a healthy adult

An autopsy case of fulminant hepatitis caused by herpes simplex virus type 1 in a healthy adult is presented. The clinical course was characterized by hepatic failure, disseminated intravascular coagulation and acute renal failure. Many small ulcerations were present in the tongue and tonsils, and there were foci of hemorrhagic necrosis in the liver. Herpes simplex viral antigen was identified in the liver, tonsils, spleen, tongue, pharynx, larynx, esophagus, stomach, intestine, adrenal glands, and lymph nodes with immunohistochemical staining using antibodies to herpes simplex virus type 1. The electron microscopic examination revealed many virions in the hepatocytes. Herpes simplex virus was isolated from the liver, and viral DNA, which had some distinctive features of herpes simplex virus type 1, was examined. We discuss possible reasons for this opportunistic infection occurring in a healthy adult.

Genomic comparison of herpes simplex virus type 1 isolates from Japan, Sweden and Kenya.

One-hundred and seventy-two epidemiologically unrelated herpes simplex virus type 1 (HSV-1) strains isolated in Japan (104 strains) and Sweden (68 strains) were compared by analysis of their genome structures using four restriction endonucleases, BamHI, KpnI, SalI and HindIII. In addition, 32 Kenyan HSV-1 isolates previously compared to Japanese isolates were included for further comparison with the Swedish isolates. Remarkable and statistically significant differences were found between the HSV-1 isolates from the three countries. One-hundred and thirty cleavage sites were examined, and it was shown that isolates from two of the three countries were statistically distinguishable at 27 of these loci. Pairwise comparison between isolates from Japan and Sweden, Kenya and Sweden, and Japan and Kenya revealed variation in 18, 16 and 23 sites, respectively. By considering gains and losses of 19 sites, the total of 204 strains could be classified into 92 distinct cleavage patterns. Isolates from the three countries could be distinguished from each other by the pattern, except for one Swedish and two Kenyan isolates which shared a pattern. Twenty-one fragments that were present or absent only in individual isolates from one or other of the three countries could be detected. These results show that HSV-1 strains from geographically separate countries or anthropologically different races have distinct distributions of endonuclease recognition sites.

A simple and practical method for typing and strain differentiation of herpes simplex virus using infected cell DNAs.

A simple and practical method for typing and strain differentiation of herpes simplex virus (HSV) isolates, based upon analysis of the restriction endonuclease cleavage patterns of viral DNAs, was established by using unlabeled infected cell DNAs. The preparation of infected cell DNA is technically easier than that of purified viral DNA or of radiolabeled viral DNA. The method provides a powerful and practical tool for epidemiological and clinical studies of HSV infection, which can be performed in most diagnostic laboratories. In order to select suitable restriction endonucleases for the study of HSV isolates, the cleavage patterns of viral DNAs (strains MacIntyre, HF, UW‐268, and SAV) with 12 enzymes were analyzed. Several enzymes, Bam HI, Kpn I, Pst I, Sal I, Sst I, and Xho I, were found to be useful for both typing and strain differentiation. With this method, HSV isolates from different individuals and from the same individual were analyzed by digestion of their infected cell DNAs with Bam HI. Six isolates from epidemiologically unrelated individuals were readily typed and differentiated from each other. Three isolates from the same individual showed very similar patterns. However, there was a small degree of difference between the first two isolates and the third isolate.

Genome variations in herpes simplex virus type 2 strains isolated in Japan and Sweden.

One-hundred and twenty-three epidemiologically unrelated strains of herpes simplex virus type 2 (HSV-2) isolated in Japan and Sweden (68 Japanese and 55 Swedish isolates) were compared by analysis of their genomes using five restriction endonucleases: BamHI, KpnI, EcoRI, HindIII and Bg/II. Seven of the 93 restriction sites examined showed statistically significant variation between isolates from the two countries. However, HSV-2 isolates were less variable than the HSV-1 isolates previously analysed from the same countries. Using 12 restriction sites as markers, the HSV-2 isolates were classified into 41 cleavage patterns; 17 were specific for Japanese isolates and 15 were specific for Swedish isolates. Correlation coefficients between some sets of 12 markers were significant, but significant correlations between Japanese and Swedish isolates were distinct for each country. Both Japanese and Swedish isolates were assigned to three major patterns with no significant difference in incidence. In contrast, in two other major patterns, differences in incidence between the isolates were statistically significant. These results suggest that HSV-2 populations in geographically separated countries have distinct cleavage site distributions.

Comparison of the association with eczema herpeticum in the two predominant genotypes of herpes simplex virus type 1

Eczema herpeticum, sometimes called Kaposis varicelliform eruption, is usually caused by a disseminated herpes simplex virus infection in a patient whose underlying skin disease is atopic dermatitis. Herpes simplex virus type 1 (HSV‐1), a widespread infectious agent in human populations, is the etiologic agent of eczema herpeticum. Analyses of restriction fragment length polymorphism (RFLP) of HSV‐1 strains isolated in Japan, using restriction endonucleases, revealed the presence of two predominant genotypes of F1 and F35. The number of HSV‐1 strains of F1 genotype was over twice that of the F35 genotype, and the nucleotide change between F1 and F35 was estimated to be 1.5%. The question of whether the genomic difference between two predominant genotypes could influence clinical manifestations remained to be addressed. On the basis of RFLP, we determined genotypes of HSV‐1 strains isolated from the patients in Japan, including those with eczema herpeticum. Two of four HSV‐1 strains of F35 genotype were from patients with eczema herpeticum, whereas none of 12 HSV‐1 strains of F1 genotype was from those with eczema herpeticum. Thus, the F35 genotype seemed to be associated more frequently with eczema herpeticum than the F1 genotype.

Detection of human papillomavirus type 2 related sequence in oral papilloma

Oral papilloma is a benign tumourous lesion. Part of this lesion is associated with human papillomavirus (HPV) infection. We analysed the genetical and histopathological evidence for HPV type 2 infection in three oral papillomas. Southern blot hybridization showed HPV 2a sequence in one lesion. Cells of the positive specimen appeared to contain high copy numbers of the viral DNA in an episomal state. In situ staining demonstrated virus capsid antigen in koilocytotic cells and surrounding cells in the hyperplastic epithelial layer. Two other specimens contained no HPV sequences by labeled probe of full length linear HPVs 2a, 6b, 11, 16, 18, 31 and 33 DNA under low stringency hybridization conditions. These results showed the possibility that HPV 2 plays a role in oral papilloma.