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Journal of Clinical Microbiology, March 2007, p. 771-782, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01236-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Contrasting Geographic Distribution Profiles of the Herpes Simplex Virus Type 1 BgOL and BgKL Variants in Japan Suggest Dispersion and Replacement{triangledown}

Hiroyuki Eda,1,2,3,§ Shigeru Ozawa,4 Kamesaburo Yoshino,4,{ddagger} Kazuo Yanagi,1,2* and the Cooperation Group for HSV-1 RFLP Variant Study{dagger}

Herpesvirus Laboratory, Department of Virus I, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan,1 Institute of Basic Medical Sciences, Tsukuba University, Tennodai 1-1-1, Tsukuba City, Ibaraki 305-8572, Japan,2 The Program of Environmental Sciences, Tsukuba University, Tsukuba, Ibaraki 305-8572, Japan,3 Yamanashi Institute of Health, Fujimi 1-7-31, Kofu City, Yamanashi Prefecture 400-0027, Japan4

Received 16 June 2006/ Returned for modification 20 August 2006/ Accepted 27 December 2006


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ABSTRACT
 
Thelifelong latent infection-reactivation mode of infection of herpes simplex virus type 1 (HSV-1) transmitted by close contact has allowed a diversity of restriction fragment length polymorphism (RFLP) variations to accumulate in human populations. Whether and how the variants of the HSV-1 that is ubiquitous worldwide spread to different human populations is not clear. In our previous study the geographically gradient distribution of the HSV-1 BgKL variant, which is a good marker for the BgKL:SaCFJM:SaGHM:SaD/EL:KpMS variant, suggested that BgKL dispersed geographically. Southern hybridization analyses showed that in BgKL the BglII cleavage site between the BglII K and small "Q/#13" fragments is lost, the SalI cleavage sites between the SalI J and C and between SalI F and J fragments are lost, and the SalI E fragment is abnormally large (SaEL variation). The RFLP and geographic distribution of one more HSV-1 RFLP variant, BgOL, were comparatively analyzed. The BglII cleavage site between the BglII O and Q/#13 fragments is lost in BgOL. BgOL clinical isolates were not associated with any of the SaCFJM, SaEL, SaGHM, or KpMS variations, whereas one-fourth of the non-BgOL:non-BgKL isolates was associated with SaCFJM and SaGHM, indicating that BgKL and BgOL are distant in terms of diversification. BgOL is distributed highly in the northeastern region and the southwestern island of Kyushu but is rare between the two regions in Japan, in a remarkable contrast to BgKL. These are the first epidemiologic data to show contrasting geographic distribution profiles of two HSV-1 variants and suggest the gradual dispersion and replacement of HSV-1 variants.


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INTRODUCTION
 
Restriction enzyme fragment length polymorphism (RFLP) is useful and widely used to differentiate the herpes simplex virus type 1 (HSV-1) isolates and strains (11, 17, 27, 35-37). The data on genetic variability based on DNA sequencing of clinical HSV-1 isolates are limited compared to other human herpesviruses (4, 5, 8, 22, 23, 41, 50). The different clustering of RFLP profiles in the HSV-1 clinical isolates in several countries and/or continents has been documented (38, 39). Intriguingly, however, the recent report by P. Norberg et al. (25) indicates that there are HSV-1 genotypic groups in HSV-1 clinical isolates from Caucasian individuals in a geographically restricted area (the western part of Sweden). HSV-1 is ubiquitous worldwide and is transmitted by human close contact (2, 47). It is not known whether and how HSV-1 variants have been replaced or are replaceable by other HSV-1 variants in human populations or geographic regions. The base sequence diversity of HSV-1 is explained by the evidence that HSV-1 diverged from HSV-2 approximately 8.4 millions years ago (19). Thus, HSV-1 arose earlier than modern humans (18, 24), cospeciated with the human host (19), and persisted in accumulating nonlethal HSV-1 variants and mutants because of the latency and reactivation mode of HSV-1 infection. HSV-1 establishes a latent infection in the ganglia in the course of primary infection, persisting for the life of the host, and recurring at the same site as the primary infection (43, 47). Thus, nonlethal HSV-1 mutants are able to stably survive in human populations. The spread of HSV-1 variants or mutants in and between human populations is slow because of its transmission mode requiring direct or close contact with mucous membranes and/or the injured skin surface (47). Such transmission requirements may hinder the penetration of HSV-1 variants into different human populations. HSV-1 is prevalent in adults and children (13, 48), and thus new HSV-1 variants must compete with existing HSV-1 viruses. However, the recent DNA sequence analyses of HSV-1 clinical isolates from Caucasian individuals in Sweden revealed that most full-length HSV-1 genomes consist of a mosaic of segments from different genetic groups (25). The frequent homologous recombination indicates that recombination is an important feature in the evolution of the HSV-1 genome and implies that an individual is reinfected with different isolates (25).

There were no published epidemiologic data that suggested geographic spread of an HSV-1 variant before our previous report (27). In a previous study, we analyzed RFLP variations of HSV-1 clinical isolates in Japan (28, 29) (Yanagi et al., Comparison of Japanese herpes simplex virus isolates by restriction enzyme fingerprinting of viral DNA, Abstr. Sixth Int. Cong. Virol., 1984, Edmonton, Canada, p. 208) and discovered the major HSV-1 RFLP variant with a BglII K fragment larger than the standard BglII K size (16); hence, designated the BgKL variant (27). The BgKL variant comprises 27.1% of the clinical isolates from patients with orolabial and skin infections (27). A great majority (91 to 97%) of the BgKL variant isolates have four other RFLP variations, SaCFJM, SaD/EL, SaGHM, and KpMS (27), making the BglII KL variation a useful RFLP marker for this cluster of five RFLP variations (27). From the geographically gradient distribution profile of the BgKL variant in terms of its relative frequencies in HSV-1 clinical isolates, we hypothesized that the BgKL variant dispersed gradually from Shikoku Island to the other Japanese Islands. People in these islands are of the same race and nationality and have long maintained stable regional communities because of the prevailing geographic, historic, and social conditions (27).

In the present study, we performed precise RFLP analyses by DNA cloning and Southern hybridization and geographic distribution analyses of the two different HSV-1 RFLP variants, BgKL and BgOL, to further investigate the hypothesized geographic dispersion of HSV-1 variants. The implications of the results that show the contrasting geographic distribution profiles of BgKL and BgOL are discussed.


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MATERIALS AND METHODS
 
Viruses and cells. Clinical specimens, cell inoculations, and identification and typing of isolates were all as described previously (27). HSV-1 viruses from viral stocks that had been prepared shortly after isolation were propagated on Vero cells in Eagle minimal essential medium supplemented with 5% calf serum in a humidified incubator containing 5% CO2 as previously described (27, 45). The HSV-1 clinical isolates examined here were obtained only from patients with HSV-1 infections of the mouth or lips or skin and included in our previous report (27), except for three clinical isolates from Oita, Nagasaki, and Miyazaki prefectures in Kyushu Island (one skin lesion isolate from each prefecture). Clinical isolates of the BglII O fragment that were too faint on agarose gel electrophoresis analyses were omitted. The laboratory strain HF was previously described (49). The standard laboratory HSV-1 strain F was kindly provided by B. Roizman (University of Chicago).

Viral DNA and restriction endonuclease cleavage analyses. DNA extraction and analyses of restriction endonuclease cleavage pattern or RFLP were performed as previously described (27). Restriction enzyme cleavage sites by theoretical digests of HSV-1 strain 17 were analyzed by using the GenBank database (NC_001806, GenBank X14112) and the REBASE database.

Cloning of BglII and SalI fragments from HSV-1 clinical isolates and laboratory strain HF and Southern blot analyses. Construction of recombinant plasmids containing restriction enzyme fragments of HSV1 DNA from clinical isolates and HSV-1 strains was carried out using the pSV2gpt vector as previously described (10). In brief, viral DNAs were extracted by using phenol and purified by CsCl density gradient centrifugation. The HSV-1 clinical isolates and strains that were used for DNA cloning were RM9 (BgKL variant, genitals, Kyushu Island), SY139 (BgKL, lips, Kyushu), RK (non-BgKL, skin, Tokyo), and the HF strain. The HSV-1 clinical isolates and strains that were analyzed by Southern blot hybridization were RM-11 (BgKL, skin, Kyushu Island), KH-250 (BgKL, skin, Tokyo), RM9, SY139, RK, F, and HF. Restriction enzyme-digested DNA fragments from the clinical isolates and strain were isolated by agarose gel electrophoresis and electroelution. Nick translation of Southern blot hybridization probes was performed by using [{alpha}-32P]dCTP (40), and the specific radioactivity of the obtained hybridization probe DNA was 1.0 x 107 to 5.0 x 108 cpm/µg. Base sequences were determined as previously described (15).

Statistical methods. The Fisher exact test was carried out by using a computer program (StatFlex version 5.0). The level of significance was P < 0.05 (two-tailed).


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RESULTS
 
Southern hybridization analyses showed that the BglII cleavage site between the BglII K fragment and a BglII fragment smaller than 0.34 kbp is lost in the BgKL variant. To examine whether a size increase mutation in the BglII K fragment is responsible for the BgKL variation, we carried out Southern hybridization analyses. Southern blot hybridization of BamHI-digested DNAs from the BgKL variant isolates KH-250 and RM 11 and laboratory strain F was performed by using a 32P-labeled normal-size BglII K fragment cloned in the recombinant DNA pHFK1 as a probe. The results revealed no difference in the size of the BamHI A, C, and A' fragments that cover the BglII K fragment entirely (Fig. 1A) (17,35) between the BgKL variant isolates and the non-BgKL clinical isolate RK or strain F (Fig. 1Ba and Bb). In addition, similar Southern hybridization analyses of the SalI-digested DNAs from the two BgKL variant isolates showed no difference in the sizes of the SalI D, N, and Y fragments, which partially overlap or are included in the BglII K fragment (17, 35) between the BgKL isolates and strain F (Fig. 1C).


Figure 1
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  		FIG. 1. Southern hybridization analyses of the unusually large BglII K fragment of the BgKL variant and a BglII fragment smaller than 0.34 kbp that is detected in strain F and non-BgKL clinical isolates but missing in isolates of the BgKL variant. (A) Restriction enzyme physical map of HSV-1 genomic DNA indicating the cleavage sites by BglII, BamHI, and SalI. The alphabetical designations of DNA fragments are indicated along the maps. Note that there are four isomeric forms of the HSV-1 genomic DNA due to the inversion of the L and S components relative to one another (16, 33), which is indicated by the bidirectional arrows at the top of the L and S components in the diagrammatic structure of HSV-1 genome. This map is based on the BglII, BamHI, and SalI cleavage maps of standard strain F DNA (17). (B) Southern blots of the BamHI-digested genomic DNAs from the BgKL variant clinical isolates KH250 and RM11 and those from the non-BgKL clinical isolate RK and strain F using the normal-size BglII K fragment cloned in the pHFK1 recombinant DNA as the hybridization probe. The detected BamHI fragments A, C (Ba), and A' (Bb) are indicated on the bottom. The band of the BamHI A is fainter than the BamHI C fragment here because the sequence of the BamHI A fragment overlapping the BglII K fragment is smaller than that of the BamHI C fragment. (C) Southern blots of SalI-digested genomic DNAs from the BgKL variant clinical isolates KH250 and RM11 and the non-BgKL strain F using the same hybridization probe as in panel B. The SalI D, N, and Y fragments that contain portions of the BglII K sequence and thus reacted with the hybridization probe are indicated at the bottom. (D) Southern blots of the BglII and BamHI double-digested DNA subfragments from the BglII K fragment. The BamHI subfragment containing the right terminal region of the BglII K fragment, denoted as the BgK^BamA fragment in panel A, taken from the BgKL variant RM9 clinical isolate, was used as a hybridization probe. The recombinant DNA containing the BglII K fragments from the BgKL variant and non-BgKL isolates are indicated at the right of the lanes; pRM9-101 and pSY139-134 contain the unusually large BglII K from the BgKL RM9 and SY139 isolates, respectively, and pHFK1 and pRK137 contain the normal-size BglII K fragment from HF strain and the non-BgKL RK isolate, respectively. (E) Southern blots of BglII-digested genomic DNA from the BgKL variant RM9 and SY139 isolates and the non-BgKL clinical isolate RK and strain F using the 32P-labeled BgKL fragment from the pRM9-101 recombinant DNA containing the RM9 isolate BgKL fragment as the probe. The sizes of the HinfII-digested fragments of pBR322 DNA are indicated at the top. The BglII fragment that is smaller than 0.34 kb is indicated as the BglII <0.34 fragment. The very small <0.34 fragment in this image was detected by long exposure. The long exposure caused the dense smear at the right of the <0.34 fragment due to the overexposed blots of larger DNA fragments. (F) Base sequence of the BglII <0.34 fragment of the genomic DNA from HSV-1 strain HF.

Next, we tested whether an extra sequence is present at the terminus of the BgKL fragment. We constructed recombinant DNAs containing the BglII KL fragments, pRM9-101 and pKH139-134, from the BgKL variant clinical isolates RM9 and KH139, respectively, and the recombinant DNA pRK137 containing the normal-size BglII K fragment from the clinical isolate RK. The four recombinant DNA clones, pRM9-101, pKH139-134, pHFK1, and pRK137, were double digested by using BglII and BamHI and examined by Southern blot assays using the 32P-labeled subfragment that contains the right terminus of the BgKL fragment, designated BgK^BamA (Fig. 1A), as the hybridization probe. The BgK^BamA fragment was prepared by BamHI and BglII double digestion of pRM9-101 DNA containing the BgKL fragment. The Southern blots of the BgK^BamA fragments from the BgKL variant isolates RM9 and KH139 are larger than those derived from strain HF and the RK isolate (Fig. 1D).

The BglII K fragments from the BgKL and non-BgKL isolates were further examined by Southern hybridization using the 32P-lableled pRM9-101 DNA containing the entire BgKL sequence as the hybridization probe. A small DNA fragment of <0.34 kbp, referred to as the BglII <0.34 fragment here, was detected in Southern blots of both the F strain and the non-BgKL clinical isolate RK, whereas the <0.34 fragment was not detected in those of the BgKL variant RM9 or KH139 isolates (Fig. 1E). The results suggest that the BglII cleavage site between the BglII K and the <0.34 fragments is lost in the BgKL variant.

Next, we analyzed the base sequence of the <0.34 fragment from strain HF (Fig. 1F). The BglII <0.34 fragment sequence is exactly the same as the sequence of the BglII smallest fragment 13 (coordinates 25148 to 25378 of HSV-1 strain 17 (NC_001806, GenBank X14112) (20, 21) and is thus referred to as BglII <0.34/#13 fragment hereafter.

The SalI cleavage sites between the SalI C and J fragments and between the SalI C and F fragments are lost (SaCFJM variation) and the SalI E, but not D, fragment is unusually large (SaEL variation) in the BgKL variant. It was found in our previous report that the four SalI fragments, i.e., the A1 fragment that is equivalent to the sum of the SalI fragments J and C, the A2 fragment that is equivalent to the sum of the SalI fragments J and F, and the SalI C and F fragments (Fig. 1A), were absent in 106 of 109 isolates (97%) of the BgKL variant (27) (Fig. 2A). It should be noted that there are four isomeric forms of the genomic HSV-1 DNA due to the inversion of the L and S components relative to one another (12, 16, 33), as indicated by the two-directional arrows of the L and S components in the diagrammatic genomic structure of HSV-1 (Fig. 2A). Therefore, both of the fragments A1 and A2 are detected as "0.5 M concentration" fragments in the genomic DNA of clinical HSV-1 isolates that are similar to strain F; note that the inversion of the S component does not result in two different-size S component-terminal fragments but only one or the SalI K fragment in the case of SalI digestion (Fig. 1A). Instead, the four unusually large fragments were present in the BgKL:SaCFJM variant isolates as shown previously (27); their sizes were equal to the sum of F and J (designated F-J); F, J, and K (designated F-J-K); J and C (designated J-C); and C, J, and K (designated C-J-K) (Fig. 1A), and the intensity of these bands are detected at a "0.25 M concentration" compared to the fragments from the unique region (Fig. 2A).


Figure 2
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  		FIG. 2. Southern hybridization analyses of the SalI cleavage profile of genomic DNA from clinical isolates of the BgKL:SaCFJM:SaD/EL variant. (A) SalI digestion profiles of genomic DNAs from clinical isolates of the BgKL variant. (B) Southern blots of SalI fragments of genomic DNAs from the BgKL variant KH250 and RM11 clinical isolates and the non-BgKL RK isolate and strain F using the BamHI E fragment from strain F (Fig. 1A) as the hybridization probe. (C) Southern blots of the SalI fragments of genomic DNA from the BgKL variant RM11 and KH250 isolates and the non-BgKL RK isolate and strain F using the BamHI L fragment from strain F (Fig. 1A) as the hybridization probe.

The Southern blots using the BamHI E fragment (F strain) that spans the SalI cleavage site between the SalI fragments J and C as the hybridization probe showed that the J and C fragments are contained in these unusually large fragments in SalI-digested DNAs from the BgKL KH250 and RM11 isolates (Fig. 2B). The results indicate that the SalI cleavage sites between the SalI J and C fragments and between the SalI F and J fragments (Fig. 1A) are lost, resulting in the appearance of the new SalI fragments, F-J, F-J-K, J-C, and C-J-K in the BgKL variant isolates.

The SaD/EL variation that is associated with all of the BgKL:SaCFJM variant isolates (27) was also analyzed by Southern hybridization. The data shown in Fig. 1C indicate that there is no difference in the SalI D fragment size between the BgKL variants and F strain, although the SalI D fragment partially overlaps the BglII K fragment. DNAs from the HSV-1 clinical isolates of the SaD/EL variant RM11 and KH250 and the non-SaD/EL RK isolate and strain F were digested with SalI and analyzed by Southern hybridization using the BamHI L fragment from the F strain as probe, since the BamHI L fragment overlaps the SalI E fragment (Fig. 1A). The results indicate that the SalI E fragments from the BgKL isolates KH250 and RM11 are larger than those from the F strain and the RK isolate (Fig. 2C).

Southern hybridization analyses of an HSV-1 RFLP variant with the unusually large BglII O fragment, designated BgOL. Another RFLP variant of HSV-1 with the unusually large BglII O fragment variant was recognized in our large-scale epidemiologic study (Fig. 3A) and was designated the BgOL variant. The present report is the first to describe the BgOL variant to our knowledge. Fifteen in vitro passages of the BgOL isolate OSU413 (Osaka Prefecture, the Kinki Region), starting from the first passage ampoule that was stocked immediately after isolation, did not change the cleavage profiles using BglII, KpnI, and EcoRI (data not shown), indicating that the RFLP characteristics of the BgOL variant are stable under these experimental conditions. The nationwide isolation frequency of the BgOL was 27 of 597 clinical isolates (4.5%).


Figure 3
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  		FIG. 3. RFLP variant BgOL with an unusually large BglII O fragment. (A) Restriction endonuclease cleavage profiles of genomic DNA from clinical HSV-1 isolates using BglII, SalI, and KpnI. The restriction enzymes are indicated at the bottom of each image. The clinical isolate (place of isolation, variation) of the HSV-1 DNA in each lane was as follows: lane 1, KT527 (Tokyo, BgOL); lane 2, FO-33 (Fukuoka, Kyushu, non-BgOL:non-BgKL); lane 3, OS420 (Osaka, Kinki, BgKL); lane 4, Lambda phage DNA digested with HindIII; lane 5, FO-57 (Fukuoka, Kyushu, BgOL); lane 6, IW30 (Iwate, Tohoku, BgOL); lane 7, RK (Tokyo, non-BgOL:non-BgKL); and lane 8, F strain (United States). The designations of DNA fragments from the F strain are marked at the right of each gel. (B) DNA cleavage profiles of the HSV-1 BgOL variant clinical isolate IW30 (one of the HSV-1 BgOL clinical isolates shown in panel A) using the restriction enzymes BglII, SalI, KpnI, BamHI, HindIII, EcoRI, HpaI, and XbaI in parallel with the non-BgOL:non-BgKL clinical isolate RK and strain F. Lane 1, RK; lane 2, IW30; lane 3, F. (C) Southern blots of BglII-digested genomic DNA from the BgOL variant clinical isolate IW12. The clinical isolate IW12 was analyzed by Southern hybridization using the same hybridization probe as that used in Fig. 1E in parallel with RK (non-BgOL:non-BgKL) and strain F. Note that the 32P-labeled BgKL fragment probe from the pRM9-101 recombinant DNA contains the BglII <0.34 fragment sequence.

Southern hybridization analyses of genomic DNAs of the BgOL variant isolate IW12 and strain F and the non-BgKL:non-BgOL isolate RK was performed in parallel to see whether the above-described BglII <0.34/#13 fragment is contained in the BgOL fragment. The Southern hybridization using the BglII KL fragment from the RM9 isolate as the hybridization probe detected the BgOL bands of IW12 (Fig. 3C) but not the normal-size BglII O bands from the F strain or the RK isolate. These results indicate that the BgOL fragment contains the BglII <0.34/#13 fragment, whereas the normal-size BglII O fragment does not, indicating that the BglII cleavage site between the BglII O and <0.34/#13 fragments is lost in the BgOL variant.

RFLP analyses with SalI and KpnI revealed that clinical isolates of the BgOL variant are not associated with any of the SaEL, SaCFJM, SaGHM, or KpMS variations that are all associated with the great majority of clinical isolates characterized by the BgKL variation. We have previously reported that the SaCFJM variation is associated with the BgKL variant at the extremely high frequency of 97.2% (106 of 109 clinical isolates), the SaEL variation is associated with the BgKL variant at 98.1% (107 of 109 isolates), the SaGHM variation that lacks the SalI G and H fragments is associated with the BgKL variant at 90.8% (99 of 109), and 89.9% (98 of 109) of the BgKL clinical isolates were BgKL:SaCFJM:SaD/EL:SaGHM (27). In addition, the KpMS variation that lacks the KpnI M fragment also is associated with the BgKL variant at the frequency of 96.3% (52 of 54 isolates) (27). In the present report, to further characterize the RFLP of the BgOL variant we cleaved DNA from BgOL variant clinical isolates using SalI and KpnI. The SalI and KpnI cleavage profiles of DNA from the three clinical isolates—KT527, FO-57, and IW30—are shown in Fig. 3A. Further DNA cleavage data on the BgOL variant IW30, in parallel with strain F and the non-BgOL:non-BgKL variant RK isolate, was obtained by using the restriction enzymes SalI, KpnI, BamHI, HindIII, EcoRI, HpaI, and XbaI and are presented in Fig. 3B. None of the SaCFJM, SaGHM, SaEL, and KpMS variations was associated with any of the BgOL isolates (0 of 17 [0.0%] isolates, 9 from the mouth or lips and 8 from the skin lesions). The differences in the association rates of the SaCFJM, SaGHM, SaEL, and KpMS variations are all statistically significant between the BgOL and BgKL variants (P < 0.0001).

Remarkably, the frequency of association of the SaCFJM variation with the BgOL variant (0.0%) is significantly lower than that with non-BgOL:non-BgKL isolates (24.1%) (P = 0.0242), and the association frequency of the SaGHM variation with the BgOL variant (0.0%) is significantly lower than that with non-BgOL:non-BgKL isolates (23.2%) (P = 0.0242) (Table 1) . For comparison, the frequency of the SaEL variation in the BgOL variant isolates (0.0%) is not significantly lower than in non-BgOL:non-BgKL isolates (0.0%) (P = 1.000), and also there is no statistically significant difference in the frequency of KpMS variation between the BgOL variant (0.0%) and non-BgOL:non-BgKL isolates (7.8%) (P = 0.5786) (Table 1).


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  		TABLE 1. Association of the BgOL and non-BgOL:non-BgKL clinical isolates with the SalI RFLP variations SaCFJM, SaGHM, and SaEL and with the KpnI RFLP variation KpMS

The geographic distribution of the BgOL variant is contrary to that of the BgKL variant and different from non-BgOL:non-BgKL isolates. To compare the geographic distribution of the BgOL variant with that of the BgKL variant, we analyzed relative frequencies of the BgOL variant in HSV-1 clinical isolates in the Tohoku, Chubu, Kinki, Chugoku, Shikoku, Kyushu, Okinawa, and Tokyo regions (Fig. 4A). The relative frequency of the BgOL is high in Tohoku, Chubu, and Kyushu and low in Shikoku-Chugoku and Okinawa (Fig. 4A and Table 2). The differences in the relative BgOL frequency between Tohoku and Shikoku-Chugoku (P = 0.0015), between Chubu and Shikoku-Chugoku (P = 0.0108), and between Shikoku-Chugoku and Kyushu (P = 0.0080) are statistically significant. The statistical significance of the differences between Tohoku and Kinki (P = 0.0506) and between Tohoku-Chubu and Kinki (P = 0.0599) are not clear, and this is indicated by the broken line between Kinki and Tohoku-Chubu (Fig. 4B). The differences in the relative BgOL frequency between Kinki and Shikoku-Chugoku (P = 0.1987) and between the Kinki and Kyushu (P = 0.1431) are not significantly different. The difference in the relative BgOL frequency between Tohoku-Chubu and Shikoku-Chugoku-Kinki is statistically significant (P < 0.001). The difference in the relative BgOL frequency between Kyushu and Okinawa (P = 0.7013) and between Kyushu and Tohoku-Chubu (P = 0.7595) are not significantly different (Table 3). The differences are not statistically significant between Okinawa and Kyushu-Kinki-Chubu-Tohoku (P = 0.5510) and between Okinawa and Shikoku-Chugoku-Kinki (P = 0.8567). The statistically significant differences in the relative frequencies of the BgOL variant between the geographic areas are summarized in Table 3 and diagrammatically depicted in Fig. 4B.


Figure 4
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  		FIG. 4. Geographic locations of the regions where the relative frequencies of the BgOL variant were examined and diagrammatic maps indicating the statistically significant differences in the frequency levels of the BgOL variant and non-BgOL:non-BgKL virus in the respective geographic regions. (A) Geographic locations of the Tohoku, Chubu, Kinki, and Chugoku regions on Honshu Island and Shikoku, Kyushu, and the Okinawa islands in Japan where clinical HSV-1 isolates were collected for the studies reported here. The numbers in parentheses indicate the percentages of the HSV-1 BgOL RFLP variant determined in the present study. The gray-and-white symbol of Okinawa Island indicates that there are no statistically significant differences between Okinawa and Kyushu or between Okinawa and Shikoku-Chugoku-Kinki. (B) A diagrammatic map representing the statistically significant difference in relative frequencies of the BgOL variant between the different geographic areas. The area where the relative frequency of the BgOL variant is high is indicated in gray, and the area where the relative frequency of the BgOL variant is rare is indicated in white. The broken line indicates that the BgOL frequency difference between the Kinki region and the Tohoku-Chubu area (A) is not clear statistically, whereas that between the Shikoku-Chugoku-Kinki and Tohoku-Chubu areas is statistically significant (Table 3). (C) Diagrammatic map representing the statistically significant difference in the relative frequencies of the non-BgOL:non-BgKL clinical isolates in the different geographic areas. The area where the relative frequency of non-BgOL:non-BgKL isolate is high is indicated in gray, and the area where the relative frequency of non-BgOL:non-BgKL isolate is not high is indicated in white. The solid line indicates the non-BgOL:non-BgKL frequency differences not only between the Shikoku-Chugoku-Kinki and Tohoku-Chubu areas but also between the Kinki Region and the Tohoku-Chubu area (A) are statistically significant (Table 4).


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  		TABLE 2. Geographic distribution of the HSV-1 RFLP variant BgOL and non-BgOL:non-BgKL clinical isolates in Japan


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  		TABLE 3. Statistical significance of the differences in the relative frequencies of variant BgOL between the geographic regions

For comparison, the differences in the relative BgKL frequency (Table 2) between Tohoku and Shikoku-Chugoku (P < 0.0001), Chubu and Shikoku-Chugoku (P < 0.0001), Shikoku-Chugoku and Kyushu (P = 0.0473), and Shikoku-Chugoku and Okinawa (P = 0.0241) are statistically significant. The difference in the relative BgKL frequency between Kyushu and the Okinawa islands is not significantly different (P = 0.0942). The difference between Tohoku and Kinki is significantly different (P < 0.0001), but there is no significant difference between Shikoku-Chugoku and Kinki (P = 0.3556).

The geographic distribution of the non-BgOL:non-BgKL viruses was also analyzed. The relative frequencies of non-BgOL:non-BgKL viruses were high in Tohoku, Chubu, and Okinawa, but low in Kinki, Shikoku-Chugoku, and Kyushu (Table 2). The differences in the relative non-BgOL:non-BgKL frequencies between Tohoku and Shikoku-Chugoku (P < 0.0001), as well as between Chubu and Shikoku-Chugoku (P < 0.0001), are statistically significant. Furthermore, the differences in the relative non-BgOL:non-BgKL frequencies between Tohoku-Chubu and Kinki (P < 0.0001) and between Tohoku and Kinki (P < 0.0001) are significantly different, which is indicated by the solid line between Kinki and Tohoku-Chubu (Fig. 4C), in contrast to the BgOL variant. The relative non-BgOL:non-BgKL frequency differences are not significantly different between Kinki and Shikoku-Chugoku (P = 0.4635), between Shikoku-Chugoku and Kyushu (P = 0.2768), or between Okinawa and Tohoku-Chubu (P = 0.2664), in contrast to the BgOL variant. The relative non-BgOL:non-BgKL frequencies between Kyushu and Tohoku-Chubu are significantly different (P < 0.0001), also in contrast to the BgOL variant. The difference in the relative non-BgOL:non-BgKL frequencies between Kyushu and Okinawa is not significantly different (P = 0.5434) but is significantly different between Okinawa and the Kyushu-Chugoku-Shikoku-Kinki area (P = 0.0347). The statistically significant differences in relative frequencies of the non-BgOL:non-BgKL clinical isolates between these geographic regions or areas are summarized in Table 4 and schematically depicted in Fig. 4C.


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  		TABLE 4. Statistical significance of the differences in the relative frequencies of non-BgOL:non-BgKL between the geographic regions


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DISCUSSION
 
In the present study, the RFLP characteristics and geographic distribution of the HSV-1 RFLP variant BgOL have been analyzed in comparison to those of the HSV-1RFLP variant BgKL. The major findings in this report are as follows.

First, in the BgKL variation the BglII cleavage site between the BglII K fragment (genomic residues 14588 to 25147, 10,560 bp, in the genomic DNA from strain 17; NC_001806, GenBank X14112) and the BglII <0.34/#13 fragment is lost. The very small BglII fragment of 231 bp was not experimentally observed in strain 17 (6, 30) or strain F (35), whereas the small BglII fragment "Q" with the length of 0.35 MDa was depicted by Locker and Frenkel (17) in the BglII physical maps of genomic DNA from strains Justin and F. In the genomic DNA sequence data of the HSV-1 strain 17 by McGeoch et al. (20), a stretch of 231 bp the base sequence of which is the same as our BglII fragment <0.34 is listed as the BglII #13 fragment by the theoretical BglII digestion using the REBASE computer program (46). Thus, the BglII <0.34/#13 fragment is referred to as the BglII Q/#13 fragment hereafter (Fig. 1A). The SaCFJM variation is due to the loss of the SalI cleavage sites between the SalI C and J fragments and between F and J in the repeated sequence "b" (Fig. 1A). Also, the SaD/EL variation (27) is because the SalI E fragment is larger than the SalI E fragment of strain F and is thus referred to here as the SaEL variation. These three mutations defined by Southern hybridization here and the SaGHM variation (27) at different sites in the genomic DNA that are associated together with the BgKL variant suggest that the BgKL variant is a unique HSV-1 variant.

Second, 4.5% of the HSV-1 clinical isolates from Japan were the BgOL variant, in which the BglII cleavage site between the BglII O and Q/#13 fragment is lost. In contrast to the BgKL isolates, BgOL isolates have none of these SalI and KpnI RFLP variations (Table 1). The extremely high associations of BgKL with the SaEL, SaCFJM, and SaGHM (89.9%) and also with the KpMS variations (96.3%), along with our previous finding that there are no statistically significant differences in the frequency of association of BgKL with SaEL, SaCFJM, SaGHM, or KpMS between the different geographic regions of Kyushu, Chugoku, Shikoku, Osaka-Shiga, Chubu-Tohoku, and Tokyo (27), suggestthat the BgKL, SaEL, SaCFJM, SaGHM, and KpMS variations appeared together, not sequentially, in Japan. The BgOL and BgKL variations were not seen together in any of the HSV-1 clinical isolates, suggesting that the mutational frequencies of these two variations are small or that the BgOL:BgKL double variants, if any, failed to survive and disperse. There is a statistically significant difference in the association with the SaCFJM and SaGHM variations between the BgOL variant and non-BgOL:non-BgKL isolates, approximately one-fourth of which are associated with these two SalI variations (Table 1). The data indicate that the BgOL variant is not divergent from the majority of HSV-1 clinical isolates represented by the laboratory strains F and HF to the degree of the BgKL:SaEL:SaCFJM:SaGHM:KpMS variant.

Third, the relative BgOL frequency is high in the Tohoku-Chubu area and Kyushu Island, especially in Tohoku, but rare in the Shikoku-Chugoku-Kinki area, especially in Shikoku-Chugoku. This geographic distribution of the BgOL variant forms a striking contrast to that of the BgKL variant, the relative frequency of which is high in Shikoku and Chugoku and lowest in Tohoku (27). In addition, the difference in the relative BgOL frequency between Tohoku-Chubu and Kinki is not statistically clear (P = 0.0599) but is significant between Shikoku-Chugoku and Kyushu (P = 0.0080), in sharp contrast to the non-BgOL:non-BgKL isolates.

It should be noted that the population on the islands, Honshu, Shikoku, and Kyushu has long been stable as the country has endured as a single nation since the seventh century without interruption, and in addition Japan has been officially almost completely closed to the rest of the world from the early 17th through the late 19th century. Regional human populations and local communities were maintained stably and separately in Japan because of the mountainous terrain and legal prohibition for people to leave or change living places under the ancient social systems such as the ownership of all lands and serfs by the emperor (7th to 8th centuries), manorialism (8th to 12th centuries), and feudalism (12th to late 19th centuries). These geographic, historic, and social conditions have let the geographic distribution profiles of HSV-1 variants form and be maintained until recently in these Japanese islands.

Fourth, what does the contrast between the geographic distribution profiles of the BgOL and BgKL variants in the Japanese islands suggest? Recent analyses indicate that Japanese populations are characterized by the presence of two major clades of Y chromosomes, although the origin of the Japanese population remains controversial (9, 14, 26). The frequency of one haplogroup lineage that is considered to be related to the Yayoi Period/people is highest on Kyushu (62.3%) and lowest in Okinawa (37.8%) and is not significantly different between Shikoku (54.3%) and Chubu (55.8%), whereas the frequency of the other haplogroup lineage that is considered to be related to the Jomon Period/people is highest in Okinawa (55.6%) and lowest in Kyushu (26.4%) and Shikoku (25.7%), thus with no difference between Kyushu and Shikoku, and is low (32.8%) in Chubu (9). The geographic distribution profile of the Jomon-related haplogroup lineage seems qualitatively similar to that of the non-BgOL:non-BgKL isolates. In contrast, remarkably, both of the distribution profiles of these two haplogroups are different from those of the BgOL and BgKL HSV-1 variants. The distribution pattern differences support our virus dispersion theory of the HSV-1 variants.

Fifth, it is not known when and where the BgOL and BgKL variants appeared initially. The single BgOL variation probably appeared earlier than the multiple BgKL:SaEL:SaCFJM:SaGHM:KpMS variation, since the simple BgOL variant with none of the SaEL:SaCFJM:SaGHM:KpMS variations (Table 1) is far less divergent than the BgKL:SaEL:SaCFJM:SaGHM:KpMS variant from the laboratory strains F (Chicago) and 17 (Glasgow) to which the majority of the HSV-1 clinical isolates are similar in RFLP. Thus, presumably, BgOL was earlier than BgKL in the Japanese islands, although another possibility, that BgKL was older than BgOL and non-BgOL:nonBgKL viruses in the Japanese islands, cannot be excluded. Similarly, BgOL may be older than the fraction (7.8 to 24.1%) of the non-BgOL:nonBgKL viruses with the SaCFJM, SaGHM, or KpMS variations (Table 1).

In any event, the BgOL and BgKL variants came to the Japanese islands from the outside or arose by mutations. A less likely interpretation is that the BgOL variant started to disperse separately from the Tohoku Region and Kyushu Island, where the BgKL was already prevalent. BgOL-infected peoples may have come to Tohoku and Kyushu or BgOL may have had an advantage for dispersion over the BgKL variant. Another interpretation may be that an ancient human population infected with the BgOL variant was driven out from Shikoku and the surrounding regions and replaced by another invading human population infected with the BgKL variant from the Eurasian continent. However, Shikoku Island, where the relative BgKL frequency is the highest in Japan, was historically not where people from the Eurasian continent that lies to the west of Japan landed because Shikoku is situated at the opposite and farther side of the Japanese islands from the Eurasian Continent and faces the Pacific Ocean. Geographically, Kyushu Island, the northern end of which faces the Korean Peninsula across the Strait of Tsushima and the Strait of Korea, is closer to both Korea and China than Shikoku Island. Okinawa Island had long been an independent kingdom and active in international trade, although ethnically the Okinawan is Japanese. Still, the relative BgKL frequency is low in Kyushu and lower in Okinawa compared to Shikoku Island (27). Theoretically, it may be that another invading human population carrying BgKL reached Shikoku first from South-East Asia, but there is no archeological evidence to support this hypothesis. In addition, historically, people of Shikoku Island did not invade the other regions in Japan. Thus, it is more likely that the BgKL variant mainly spread from Shikoku Island by virus dispersal and gradually replaced the BgOL variant in the surrounding regions.

We consider that the accumulation of various mutations of HSV-1 in human populations and the dispersion of HSV-1 mutants to and from other populations played important roles in forming the distribution profiles of HSV-1 variants in Japan on the basic conditions of ancient and ancestral peoples' migration. Whichever variant, BgOL or BgKL, appeared earlier in the Japanese islands, the results in the present study are the first epidemiologic data to show contrasting geographic distribution profiles of the two HSV-1 variants and suggest the gradual dispersion and replacement of HSV-1 variants under stable social conditions without large-scale, drastic human tribal turnover. This conclusion supports the speculation that random mutations were conserved and dispersed in different populations (34), although this does not exclude the possibility that the evolution of HSV-1 is host dependent (38).

Sixth, the BgOL relative frequency is rare (0.0%) in the Shikoku-Chugoku area. Furthermore, there is a statistically significant difference in the geographic distribution profile between the BgOL variant and non-BgOL:non-BgKL viruses (Fig. 4). These data suggest that the BgOL variant was replaced not only by the BgKL variant but also by certain other non-BgOL variants as well, based on the BgOL-BgKL displacement hypothesis.

Finally, until recently it was believed that exogenous reinfection of HSV-1 was rare or very low in an immunocompetent host (3, 42). However, it has recently been suggested that exogenous reinfection of HSV-1 contributes to the emergence of new variants through recombination (25). Therefore, a new HSV-1 variant would be able to gradually replace other HSV-1 viruses existing in a human population when a new mutant or variant of HSV-1 differs in virulence-related properties, as shown previously (1, 7, 32), and acquire the advantage of certain altered biological properties over other HSV-1 strains. An increase in the frequency of recurrence or in the efficiency of transmission would enable the emergence of such an HSV-1 replacement.

The RFLP characteristics of the BgOL and BgKL variants indicate that they are distant in terms of diversification and the geographic distribution profiles of the two variants stand in remarkable contrast. The emergence of these features took place in the Japanese islands where, historically, people of the same racial and ethnic background have long continued to live, maintaining stable local and regional communities. We conclude that the BgKL variant that dispersed from Shikoku Island gradually replaced the BgOL variant. These are the first epidemiological data that suggest a replacement of HSV-1 variants by other variants. The sensitivity of the RFLP method to detect base substitutions is limited compared to the base sequencing method, but the RFLP analyses of the entire genomic DNAs of HSV-1 clinical isolates give an important clue to the overall variability and diversity of the HSV-1 genome. Polymorphism analyses of particular genes (25, 31, 44) of many clinical isolates of the BgKL and BgOL variants at the base sequence level will give important information concerning the divergence between the two variants and about clinical isolates of each variant along with their genotypic properties (25) in the future study. Also, analyses of the geographic distribution of BgKL and BgOL in various Asian and Pacific areas will give clues to their origin and age. Further studies on the genetic and biological properties of the BgKL and BgOL variants may yield a biological basis for the hypothesized variant dispersion and replacement and will contribute further to an understanding of viral diversification, dispersion, distribution, and epidemiology.


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ACKNOWLEDGMENTS
 
The names and institutional affiliations of the contributing members of the Cooperation Group for HSV-1 RFLP Variants Study are as follows (all in Japan): Kozaburo Hayashi, Public Health Research Institute of Kobe City, Kobe City, Hyogo Prefecture; Toshiyuki Funabashi, Toranomon Hospital, Minato-ku, Tokyo; Seiichiro Hata, Osaka University Medical School, Osaka City, Osaka Prefecture; Hiroki Iga, The University of Tokushima School of Dentistry, Tokushima City, Tokushima Prefecture, 770; T. Ikushima, Shizuoka Institute of Environment and Hygiene for HSV-1 Clinical Isolates from Shizuoka; Tomoo Itagaki, Shimane Prefectural Institute of Public Health and Environment Science, Matsue City, Shimane Prefecture 690-0122; Rinji Kawana, Iwate Medical University School of Medicine, Morioka City, Iwate Prefecture; Shunsaku Kobayashi, Yamaguchi University School of Medicine, Ube City, Yamaguchi Prefecture,755-8505; Yoshikatsu Ozaki, Shiga University of Medical Science, Shiga Prefecture; Ryoichi Mori, Kyushu University Faculty of Medicine, Fukuoka City, Fukuoka Prefecture 819-0395; Takashi Nakakita, Nagoya City Public Health Research Institute, Nagoya City, Aichi Prefecture 467-8615; Yoshio Numazaki, Sendai National Hospital, Sendai City, Miyagi Prefecture 983-8520; Shigeru Yamamoto, Kurume University School of Medicine, Kurume City, Fukuoka Prefecture 830-0011; and H. Yoshitake, School of Medicine, Ryukyu University, for HSV-1 clinical isolates from Okinawa.

We are grateful to Kozo Ishizuka, the Program of Environmental Sciences, Tsukuba University, for support to H.E. and to Tokuhiko Higashi, Institute of Basic Medical Sciences, Tsukuba University, Tennodai 1-1-1, Tsukuba City, Ibaraki 305-8572, for support to K.Y. Financial support for this research was provided by grants-in-aid from the Ministry of Health, Labor, and Welfare of Japan and the Yamanashi Institute of Health.

We thank M. Futamura, Aichi Prefectural Colony Hospital, for HSV-1 clinical isolates from Aichi; M. Hayashi, Hayashi Dermatology Clinic, for HSV-1 clinical isolates from Tokyo; H. Ishiko, MBC, for HSV-1 clinical isolates; M. Niimura, Jikei Medical University, for HSV-1 clinical isolates from Tokyo; T. Ogino, Hiroshima University, for HSV-1 clinical isolates from Hiroshima; H. Shioda for HSV-1 clinical isolates from Tokushima; Fukushima Medical School Hospital for HSV-1 clinical isolates from Fukushima; Kanto-Teishin Hospital for HSV-1 clinical isolates from Tokyo; Momoyama Hospital for HSV-1 clinical isolates from Osaka; National Zentuji Hospital for HSV-1 clinical isolates from Kagawa; and R. Kitamura for technical assistance.


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FOOTNOTES
 
* Corresponding author. Present address: AIDS Research Center, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku, Tokyo 162-8640, Japan. Phone: (81) 3-5285-1111. Fax: (81) 3-5285-1150. E-mail: kyanagi{at}nih.go.jp. Back

{triangledown} Published ahead of print on 10 January 2007. Back

§ Present address: Discovery Biology Research, Nagoya Laboratories, Pfizer Japan, Taketoyo 5-2, Aichi 470-2393, Japan. Back

{ddagger} Deceased after the completion of this study. Back

{dagger} Contributing members of the Cooperation Group are listed in the Acknowledgments. Back


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Journal of Clinical Microbiology, March 2007, p. 771-782, Vol. 45, No. 3
0095-1137/07/$08.00+0     doi:10.1128/JCM.01236-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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