Evolution of herpes simplex virus type 1 under herpesviral evolutionary processes.

Herpesviruses, 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 mucocutaneous 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.

Genomic heterogeneity maps to tandem repeat sequences in the herpes simplex virus type 2 UL region.

Two Bam HI Y and W fragments in the unique long sequence (UL) of the herpes simplex virus type 2 (HSV-2) were found to be heterogeneous in size among clones derived from a single strain as well as from epidemiologically unrelated isolates. More detailed restriction maps of these BamHI fragments were constructed and the heterogeneous subfragments were defined, cloned, and sequenced in order to investigate the mechanism causing the size difference. The subfragment of BamHI Y contained a tandem repeat sequence consisting of different numbers of 15 bp, 5'AGGGGCGGCTGGGGC3' as one unit among three isolates, and the subfragment of BamHI W contained the other tandem repeat sequence, 9 bp, 5'CCTCCCGCC3'. In the UL of the HSV-2 strain, these tandem repeat sequences were conserved and each repeat number appeared to be highly variable through viral genome replication. These results showed that the fragment length polymorphisms in these regions were attributable to the variation of unit numbers of the tandem repeat sequences.

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.

Identification of human papilloma virus DNA sequence in the hyperplastic epithelium of an oral denture fibroma.

The human papilloma virus (HPV) associated with hyperplastic epithelium in an oral denture fibroma was examined by southern blot hybridization. Extracted DNA was hybridized with full length linear HPV type 2a, 6b, 11, 16, 18, 31 and 33 DNAs as a mixed probe only under low stringent conditions. The hybridized bands digested with Bam HI and Eco RI were approximately 8.8 kbp and 15 kbp, respectively. Thus the lesional HPV DNA was different from HPV types used as probes and was probably integrated into host cell chromosomal DNA judging by the off-size high molecular weight bands. Considering the contaminating mesenchymal region and uninfected epithelial cells as well as the evidently limited homology with probe HPV DNAs, the virus copy number in infected cells was poorly defined. In situ antigen staining signals were widely detected in the hyperplastic epithelial layer.

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

Herpes 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 approximately 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.

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

Eczema herpeticum, sometimes called Kaposi's varicelliform eruption, is usually caused by a disseminated herpes simplex virus infection in a patient whose underlying skin disease in 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.

Demonstration of either endogenous recurrence or exogenous reinfection by restriction endonuclease cleavage analysis of herpes simplex virus from patients with recrudescent genital herpes.

By using restriction endonuclease (RE) cleavage analysis, either endogenous recurrence or exogenous reinfection of herpes simplex virus (HSV) was clarified in 45 male and 20 female subjects with recrudescent genital herpes. All of the plural (two to ten) isolates from 63 (205 isolates) out of 65 subjects (97%) were HSV-2. Two isolates from only one of 65 subjects (1.5%) were HSV-1, and they showed the same RE profile. In addition, an HSV-1 and five HSV-2 isolates were obtained from the remaining one female patient (1.5%), indicating that an exogenous HSV-1 strain has been reinfected during HSV-2 recrudescences. HSV-2 isolates were furthermore classified into genotypes of HSV-2 using 16 different RE markers with five REs. Two hundred and ten HSV-2 isolates from 64 subjects were classified into 27 distinct genotypes, in which some predominant genotypes and seven new genotypes were found. Plural HSV-2 isolates obtained from 63 out of 64 subjects, including one subject from whom an HSV-1 and five HSV-2 strains were isolated, were classified into the same genotypes, indicating that they may be regarded as recrudescent genital herpes by the reactivation of the same endogenous strain. However, the RE profiles of two HSV-2 strains from the remaining one subject were different. Thus, it was finally found that only two out of 65 subjects (3%) were reinfected with exogenous strains. These results lead to the conclusion that almost all recrudescent genital herpes are due to the reactivation of an initially infected HSV-2 strain, and are occasionally due to reinfection with distinct HSV strains of either the same or a different type.

Analysis of genomic polymorphism among herpes simplex virus type 2 isolates from four areas of Japan and three other countries.

Genomic polymorphism of 307 epidemiologically unrelated strains of herpes simplex virus type 2 (HSV-2) from four areas of Japan and three other countries (Korea, Sweden and the U.S.A.) was analysed by using 16 variable markers selected from 97 restriction endonuclease (RE) sites with five REs. In addition to the 16 markers, 26 rare variable RE sites were found in 307 isolates. Five and four of 16 markers (RE sites) were found to differ in the frequency of isolates with the markers between isolates from Japan and Sweden and between those from Japan and the U.S.A., respectively, suggesting that they are genomically different from each other. In this manner, 307 HSV-2 isolates from four countries could be classified into 68 different genotypes (no. of isolates/no. of genotypes = 4.5). Some isolates from one country or more than two countries at times were classified into the same genotypes, which were referred to as predominant genotypes. The most predominant genotypes for isolates from Japan, Sweden and the U.S.A. were genotypes 30, 26 (32) and 3, respectively, indicating that they are different by the country. In genotypes 1 and 6, the frequency of isolates was found to be significantly different between Japan and Sweden and between Japan and the U.S.A., respectively. Nine out of 16 markers differed in the most genomically distant isolates, each of which was obtained in Sweden and Japan. In addition, high correlation coefficients (r) in the Japanese isolates were detected in different pairs of markers from those in the Swedish isolates, suggesting that isolates from these two countries are evolutionarily distant.

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. A case report.

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.

Homogeneity and diversity of genome polymorphism in a set of herpes simplex virus type 1 strains classified as the same genotypic group.

Restriction fragment length polymorphism (RFLP) of 327 strains of herpes simplex virus type 1 (HSV-1) isolated in Japan were analyzed using six restriction endonucleases (REs) recognizing 6 base pairs (BamHI, HindIII, KpnI, PvuII, SalI, XhoI). The presence of strains with the same RFLP profile became evident. Fifteen strains of each of the two predominant sets with the same RFLP profile were further analyzed using two different methods, that is analyses of RFLP using 3 restriction endonucleases recognizing 4 base pairs (4-bp REs) (HaeIII, HhaI, MboI) and analyses of the variation of 3 reiterated sequences (reiterations I, IV, VII). Most of the epidemiologically unrelated strains could be differentiated by variation of the reiterations. RFLPs differentiating the strains were detected with the 4-bp REs, and epidemiologically related strains shared a specific RFLP, thereby confirming the relationship. These results verified the utility of the two methods for use in molecular epidemiological studies. Homogeneity of RFLP among the strains suggested derivation from a common ancestor (making up a genotypic group) while diversity in strains of the same genotypic group was indicative of a separate replication.

[Diagnosis of varicella-zoster virus (VZV) infection by using FITC-labeled monoclonal antibodies].

Anti-varicella zoster virus (VZV) mouse monoclonal antibodies conjugated with fluorescein isothiocyanate were evaluated for their usefulness as a practical diagnostic tool in the clinical field by examining cells infected with isolated herpes viruses and 431 clinical samples. The kit stained clearly the cells infected with 14 isolated VZV strains without cross reaction to 15 isolated herpes simplex virus type-1 strains (HSV-1) and 14 type-2 (HSV-2) strains. In clinical specimens, viral antigens of VZV were detected in 92/105 (87.6%) cases of varicella and in 176/190 (92.6%) cases of herpes zoster. Specific fluorescence of VZV was also observed in 5 out of 96 cases diagnosed as HSV infections, although these samples had no specific reaction to HSV when tested by the commercially available diagnostic kit. In 24 cases which could not be clinically diagnosed as herpes zoster or herpes simplex, the VZV antigen was demonstrated in 9 cases. All 109 VZV-positive cases in virus isolation by culture were also judged VZV-antigen positive by the kit, while all 69 HSV-positive cases in virus isolation were VZV-antigen negative. Furthermore, the VZV antigen was detected by the kit in 53/60 clinical diagnoses of varicella or herpes zoster without successful virus isolation. These results clearly indicate the usefulness of the kit as a practical VZV diagnostic reagent, especially in terms of specific sensitivity and easy technical manipulation.

A tandem repeat sequence found in a heterogeneous fragment of UL of herpes simplex virus type 1.

We found a tandem repeat sequence in the region (designated BS7) in which restriction fragments BamHI D and SalI B overlap each other, near the centre of the unique long sequence (UL) of the herpes simplex virus type 1 (HSV-1) strain F genome. The SmaI physical map of BS7 was constructed, and the position of a heterogeneous SmaI subfragment from HSV-1 isolates and plaque-purified clones from a single strain was defined on the map. The maximum size difference in the SmaI subfragment was estimated to be 600 bp between these isolates and 100 bp between the clones. The 0.23 kb SmaI subfragment recloned from BS7 was sequenced, and was shown to contain a tandem repeat sequence consisting of 15 units of 12 bp, 5' TTGGGGCTGGGG 3'. These results suggest that the fragment length heterogeneity in the UL of HSV-1 isolates and clones is attributable to copy number variation of the tandem repeat sequence.

[A clinical study of genital herpes and the clinical efficacy of acyclovir tablets].

A clinical study for genital herpes was conducted on 154 patients and the efficacy of treatment with oral acyclovir was investigated in 51 of these patients. The diagnosis was confirmed by direct immunofluorescence or viral isolation from the lesion. This disease has increased in both males and females in recent years and was found in 2.3-2.9% of the out-patients examined in 1986. Seventy percent of the patients were between 20 and 30 years old. About 70% of the male patients had phimosis. In patients with the first infection, bilateral eruption (62%) and lymphadenopathy (54%) were more common than unilateral lesions. However, in those with recurrent infection, unilateral eruption (72%) and lymphadenopathy (52%) were more common. Sixty two percent of those with the first infection had scattered eruption on external genitalia, but 71% with recurrent infection, had lesions concentrated in several areas. Local symptoms such as pain in the external genitalia (male: female, 16%: 85%), pain in the lower extremities (26%: 45%), discomfort in the lower extremities (20%: 41%) and systemic symptoms such as malaise (22%: 48%) and anorexia (4%: 35%) were seen more frequently in females than in males. In addition, systemic symptoms such as fever (first episode: recurrent episode, 36%: 4%), malaise (34%: 9%) and anorexia (18%: 2%) were seen more frequently in patients with the first episode than in those with recurrence. HSV type 1 infections were found in 16% of males and 28% of females with the first episode, but were less common in the recurrent episode, 0% and 13%, respectively. Direct immunofluorescence was positive in 75 (59%) of 128 samples diagnosed by viral isolation. Treatment with oral acyclovir tablets, 200 mg five times daily, was very effective in 26 of 30 patients (87%). No side effects were observed. In this study, acyclovir tablet has been shown to be a very effective and well-tolerated treatment for genital herpes infections.

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.

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.