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Journal of Clinical Microbiology, June 2006, p. 2109-2118, Vol. 44, No. 6
0095-1137/06/$08.00+0     doi:10.1128/JCM.02064-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Geographical Distribution of the Herpes Simplex Virus Type 1 BgKL Variant in Japan Suggests Gradual Dispersion of the Virus from Shikoku Island to the Other Islands

Shigeru Ozawa,1 Hiroyuki Eda,2,3 Kozaburo Hayashi,4 the Cooperation Group for HSV-1 RFLP Variant Study, Kamesaburo Yoshino,1,{dagger} and Kazuo Yanagi2*

Yamanashi Institute of Health, Fujimi 1-7-31, Kofu, Yamanashi 400-0027, Japan,1 Herpesvirus Laboratory, Department of Virology I, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan,2 The Master's Program of Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan,3 Public Health Research Institute of Kobe City, Kobe City, Hyogo Prefecture 650-0046, Japan4

Received 3 October 2005/ Returned for modification 1 January 2006/ Accepted 6 March 2006


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ABSTRACT
 
Restriction endonuclease fragment length polymorphism (RFLP) is useful for the epidemiological study of herpes simplex virus type 1 (HSV-1). We report here the identification of a major BglII RFLP variant of HSV-1, designated BgKL, found in 27.0% of 636 HSV-1 clinical isolates. We have also established its geographic distribution in Japan. BgKL has an unusually large BglII K fragment. SalI cleavage analyses showed that 97% of BgKL variant isolates lack both the SalI C-J and the F-J cleavage sites and have an unusually large SalI D or E fragment, and 91% of the BgKL variants lack both SalI G and H fragments. Furthermore, 96% of BgKL isolates have an unusually small KpnI M fragment. Therefore, BgKL is a marker for these five mutations in most HSV-1 isolates and is a useful HSV-1 RFLP marker. The BgKL variant was found in 59% of HSV-1 isolates from Shikoku Island, 44% of HSV-1 isolates from the Chugoku region of Honshu Island, 31% of HSV-1 isolates from Kyushu Island, 0% of HSV-1 isolates from Okinawa Island, 49% of HSV-1 isolates from Osaka, 27% of HSV-1 isolates from Shiga, 13% of HSV-1 isolates from the Chubu Region, and 9% of HSV-1 isolates from the Tohoku Region of Honshu Island. Differences in the frequency of BgKL between the Shikoku-Chugoku-Osaka area (49%) and Kyushu, between Kyushu and Okinawa, between the Shikoku-Chugoku-Osaka area and Shiga, and between Shiga and Tohoku are all statistically significant. The BgKL frequency decreases in a geographical gradient suggest that this HSV-1 variant was dispersed from Shikoku to the surrounding regions and then to more distant regions. The BgKL frequency in Tokyo was similar to the nationwide average. These are the first data to suggest a geographic and demographic dispersion pattern of HSV-1. Implications for the epidemiology and diversification of HSV-1 are discussed.


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INTRODUCTION
 
DNA restriction endonuclease cleavage patterns and restriction fragment length polymorphism (RFLP) are useful for epidemiological studies of herpes simplex virus type 1 (HSV-1) strains (7, 14, 15, 25, 28, 29). RFLP profiles have been used to differentiate HSV-1 clinical isolates from epidemiologically unrelated individuals and to determine whether viruses isolated from epidemiologically "connected" individuals are identical (1-3, 5, 9, 12, 13, 16, 18, 19, 23, 26, 28, 36). The data indicate that the genomic DNAs of HSV-1 isolates have accumulated enough base substitutions to allow different isolates to be distinguished. HSV-1 polymorphism also has been observed by differences in the electrophoretic patterns of virion structural proteins of different HSV-1 strains (17, 21, 28). The lifelong latency and reactivation of HSV-1 infection probably facilitates the accumulation of random mutations and diversification of HSV-1 (25, 37, 38). HSV-1 isolates with distinct base substitution mutation(s) at restriction endonuclease cleavage sites are designated RFLP variants in here.

It is not known whether an HSV-1 RFLP variant isolated at a certain frequency in a particular population is due to an RFLP variation that arose in that population or that migrated in from a different outside population. Analyses of the geographic distributions of distinct RFLP variants in neighboring geographic regions or populations in which many generations have lived continually in their birthplace communities should provide data on the origins of the variants because HSV-1 transmission is by intimate contact between individuals. Japan is suitable for such a geographical study because it was almost completely closed to the rest of the world for two and a half centuries before the late 19th century. Furthermore, people were obliged to live within their birthplace neighborhood by their ancient social systems for more than 13 centuries. In addition, until recently, the mountainous terrain on the Japanese islands kept local communities stable.

In the present study we analyzed 636 clinical HSV-1 isolates from 16 prefectures on the four Japanese islands (Honshu, Shikoku, Kyushu, and Okinawa) where Japanese have lived continuously since prehistoric times and, for comparison, from the Tokyo metropolitan area into which many people have moved from throughout Japan.


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MATERIALS AND METHODS
 
Viruses and cells. All HSV-1 viruses used in the present study were isolated from patients with orolabial/oropharyngeal disease (e.g., labialis, gingivostomatitis, pharyngitis, and sore throat) or patients with vesicular or ulcerative lesions on the skin or eyelid, including eczema herpeticum (37, 38). The average age of the patients was 14 years (range, 0 to 79 years). Clinical specimens, cell inoculations, and identification and typing of isolates were described previously (35). Briefly, sterile swabs soaked in distilled water containing penicillin and streptomycin were applied to the lesion and then put into 2.0 ml of the transport medium (Earle's balanced salt solution containing 0.5% lactalbumin hydrolysate, 0.1% yeast extract, and 20% inactivated normal calf serum). A total of 0.2 ml of the specimen was inoculated into Vero cell cultures, followed by incubation at 37°C with daily observation of cytopathic effects (CPE). When the CPE were clearly distinguishable, the fluid was harvested and stored at –70°C. Virus identification was performed using HSV-1 antiserum obtained by hyperimmunizing a rabbit with the HF strain and heat inactivated at 56°C for 30 min.

The standard laboratory HSV-1 strain F was kindly provided by B. Roizman (University of Chicago). HSV-1 viruses, from virus stocks that had been prepared shortly after isolation, were propagated on Vero cells (42, 43) in Eagle minimal essential medium supplemented with 5% calf serum in a humidified incubator containing 5% CO2 (35, 39, 40, 45).

The geographic distribution of the 636 HSV-1 isolates used in these studies is shown in Fig. 1 and Table 1: (i) 44 isolates from Shikoku Island (40 from Tokushima Prefecture and 4 from Kagawa Prefecture), (ii) 99 isolates from Fukuoka Prefecture on Kyushu Island, (iii) 14 isolates from Okinawa Island, (iv) 81 isolates from the Chugoku Region (21 from Yamaguchi Prefecture, 19 from Hiroshima Prefecture, and 41 from Shimane Prefecture), (v) 86 isolates from the Kinki Region (33 from Shiga Prefecture and 53 from Osaka Prefecture), (vi) 112 isolates from the Chubu Region (60 from Aichi Prefecture, 7 from Shizuoka Prefecture, and 45 from Yamanashi Prefecture), (vii) 98 isolates from the Tohoku Region (17 from Fukushima Prefecture, 45 from Miyagi Prefecture, and 36 from Iwate Prefecture), and (viii) 102 isolates from the Tokyo metropolitan area.


Figure 1
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  		FIG. 1. Geographic locations of the areas of the 16 prefectures on Honshu, Shikoku, Kyushu, and 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 BgKL RFLP variant determined in the present study.


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  		TABLE 1. Geographic distribution of the HSV-1 RFLP variant BgKL in Japan

Except for seven isolates from Shizuoka Prefecture that were obtained in 1987, all of the clinical isolates were pre-1985, when the populations in the other areas, except Tokyo, remained much more stable than they have since then.

Virus DNAs and restriction endonuclease cleavage analyses. Vero cells were infected at a multiplicity of infection of ~0.1. Infected cells were freeze-thawed and clarified by a low-speed centrifugation. The supernatant was centrifuged at 15,000 rpm (27,000 x g) for 1 h, and the pellet was suspended in phosphate-buffered saline and then incubated with 0.25 mg of proteinase K (Seikagaku Kogyo, Tokyo, Japan)/ml at 37°C for 30 min in the presence of 0.25% sodium dodecyl sulfate and 1 mM EDTA in Tris-HCl buffer (pH 7.8). DNAs were extracted with phenol and chloroform. DNAs were digested with restriction endonucleases (from Takara Shuzo, Japan, and New England Biolabs, Massachusetts) and analyzed by gel electrophoresis in 0.8% agarose (Seakem ME; FMC, Rockland, Maine) slab gels in 50 mM Tris-HCl buffer (pH 7.8) containing 2 mM EDTA and 20 mM sodium acetate.

Statistical methods. P values were calculated by using the Fisher exact test and are given in parentheses in the text, unless otherwise indicated.


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RESULTS
 
RFLP analyses of HSV-1 clinical isolates using the restriction endonuclease BglII. For the analysis of HSV-1 clinical isolates, restriction endonuclease BglII was used in the present study because the number (12 major fragments) and sizes of HSV-1 BglII fragments facilitated their separation and identification by agarose gel electrophoresis (14, 24, 28). Many HSV-1 isolates were found to have an RFLP variation characterized by a BglII K fragment larger than that of HSV-1 strain F (Fig. 2A, lanes 2, 4, 5, and 7, and B). This RFLP variation is designated BgKL in the present study. The lengths of 11 other BglII fragments of the BgKL variant DNAs were identical to those of strain F DNA (Fig. 2A, lanes 2, 4, 5, and 7). The BgKL variant was identified in 172 of the 636 (27.0%) HSV-1 isolates in these studies. To our knowledge, this is the first report of the BgKL variant. Because of the high frequency of BgK variants, the HSV-1 isolates were analyzed with other restriction enzymes.


Figure 2
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  		FIG. 2. (A) Restriction endonuclease BglII, SalI, and KpnI cleavage profiles of DNAs from seven clinical HSV-1 isolates. The restriction enzymes used are indicated at the bottom of each gel. The clinical isolates (place of isolation) of the HSV-1 DNA in each lane are as follows: lane 1, RK (Tokyo); lane 2, IW20 (Iwate); lane 3, IW51 (Iwate); lane 4, FO-48 (Fukuoka); lane 5, FS467 (Fukushima); lane 6, TS64 (Tokushima); lane 7, TS108 (Tokushima); lane 8, F strain (United States). The designations of the HSV-1 F strain DNA fragments are marked at the right of each gel. (B) Physical map of the restriction endonuclease fragments of HSV-1 DNA showing the BglII and SalI fragments. The heavy line in the BglII map denotes the BglII K fragment. The KpnI map shows only the KpnI M fragment. The genomic structure of HSV-1 DNA is schematically depicted in the top two maps. The L and S components, the unique regions UL and US, and the terminal repeat sequences "ab" and "ca" are marked. The terminal sequences "a," "b," and "c" are inversely repeated at the junction of the L and S components. The L and S components invert relative to each other, producing four HSV-1 isomeric DNAs (8, 22, 30).

RFLP analysis of HSV-1 BgKL variants using the restriction endonuclease SalI. SalI was used for these RFLP analyses because a SalI physical map of HSV-1 strain F has been reported in combination with its BglII physical map (14). The RFLP studies of BgKL isolates, including TS108 from Tokushima, Shikoku, identified isolates that had lost the SalI C fragment containing a part of the terminal "b" repeat and the SalI F fragment containing a part of the internal inverted "b" repeat, and the SalI J and K terminal fragments (Fig. 3). It is noteworthy that the concentrations of the SalI J and K terminal fragments are known to be half-molar relative to the SalI fragments from the unique regions because of the isomeric structure of HSV-1 DNA (8, 22, 34) (Fig. 3). All of the BgKL isolates that had lost SalI J and K terminal fragments had four new large fragments, with sizes corresponding to the combined sizes of fragments F and J, fragments C and J, fragments F, J, and K, and fragments C, J, and K, indicating that they are fragments F-J, C-J, F-J-K, and C-J-K, respectively (Fig. 2 and 3). This SalI RFLP variation is designated SaCFJM here. The SaCFJM variation indicates that the SalI cleavage sites between the J and C fragments and between the F and J fragments have been lost. The SaCFJM variation was detected in 106 of 109 (97.2%) BgKL isolates (Table 2). The double mutant with BgKL and SaCFJM is designated a BgKL:SaCFJM variant.


Figure 3
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  		FIG. 3. DNA cleavage profiles of HSV-1 strains using BglII, SalI, KpnI, BamHI, HindIII, EcoRI, HpaI, and XbaI. Strains TS108 (an HSV-1 BgKL clinical isolate from Tokushima), RK (a non-BgKL HSV-1 clinical isolate from the Tokyo metropolitan area), and F (HSV-1 strain F) were analyzed in parallel gels. Lane 1, RK; lane 2, TS108; lane 3, F.


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  		TABLE 2. Association of the SalI RFLP variations SaCFJM, SaD/EL and SaGHM with the BgKL variation

Surprisingly, all BgKL:SaCFJM isolates also had a slightly larger SalI D or E fragment from the UL region than the F strain (Fig. 2 and 3 and Table 2); isolates with a larger D or E fragment are designated SaD/EL variants here. BgKL isolates also having SaCFJM and SaD/EL variations are designated BgKL:SaCFJM:SaD/EL. Furthermore, 92.5% of the BgKL:SaCFJM:SaD/EL isolates lacked both SalI fragment G from the US region and H fragment from the UL region (Fig. 2 and 3 and Table 2). Isolates that have lost the G and H fragments are designated SaGHM variant here, and BgKL isolates that are also SaGHM are designated BgKL:SaGHM variants; 90.8% of the BgKL isolates were BgKL:SaGHM variants. These data show that most (89.9%) BgKL variants had the BgKL:SaCFJM:SaD/EL:SaGHM variation (Table 2).

Of the three BgKL variants without the SaCFJM variation, one isolate (TK509 from Tokyo) had the SaD/EL variation, another (OS366 from Osaka) had the SaGHM variation, and the third (Y79-98 from Yamanashi) had neither the SaCFJM nor the SaD/ELvariation (Table 2).

We also analyzed the frequencies of SalI RFLP variations in non-BgKL clinical isolates. The SaCFJM variation was detected in 23.3% non-BgKL isolates, which is significantly lower than its frequency (97.2%) in BgKL isolates (P < 0.001). Only one clinical isolate, RM45 from Kyushu, of the 52 SaCFJM variants with a normal size Bgl II K fragment had the SaD/EL variation (Table 2), in contrast to 100% of the BgKL SaCFJM isolates. None of the remaining 171 non-BgKL isolates without the SaCFJM variation had the SaD/EL variation (Table 2), indicating that SaD/EL variation is very rare in non-BgKL isolates, in contrast to BgKL variants. The SaGHM variation was detected in 22.0% non-BgKL isolates (Table 2). The difference between BgKL and non-BgKL isolates in the frequency of the SaGHM variation is statistically significant (P < 0.001).

These results, taken together, indicate that the SaCFJM, SaD/EL, and SaGHM variations are common in the majority of BgKL isolates but not in non-BgKL isolates.

RFLP analysis of HSV-1 BgKL variants using restriction endonuclease KpnI. We further characterized BgKL variants using KpnI because the KpnI cleavage map of the HSV-1 F strain has been reported (14). Of the BgKL variants studied, 96.3% had lost the KpnI M fragment found in the F strain and instead had a truncated fragment of 4.1 kb between the P and Q/R fragments (Fig. 2 and 3 and Table 3). This KpnI RFLP variation is designated the KpMS variation in the present study. In contrast, only 9.0% non-BgKL isolates had the KpMS variation. These results indicate that the frequency of the KpMS variation is much higher in BgKL isolates than in non-BgKL isolates (P < 0.001), and the majority of BgKL isolates are BgKL:KpMS.


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  		TABLE 3. Association of the KpnI RFLP variation KpMS with the BgKL variation

Geographical distribution of HSV-1 BgKL variants. The very high coincidence of BgKL, SaCFJM, SaD/EL, and KpMS RFLP variations in the same isolate indicates that the BgKL variant is a distinctive RFLP variant and useful as a marker of this set of concomitant mutations. Therefore, we carried out large-scale analyses of the geographic distribution of BgKL on four major islands of Japan (Fig. 1). The results of these BgKL prevalence analyses in different geographic regions were as follows.

Of the isolates from Tokushima, Shikoku Island, 60.0% were BgKL variants, and of those from Kagawa, Shikoku, two of four were BgKL variants. The BgKL frequency, 59.1%, on Shikoku Island is significantly higher than the nationwide average (P = 0.00002) and is the highest of all of the regions examined (Table 1). Of the isolates from Osaka, 49.1% were BgKL variants (Table 1). Of the isolates from the Chugoku region, 44.4% were BgKL variants; in particular, 47.4% were from Hiroshima, 46.3% were from Shimane, and 38.1% were from Yamaguchi (Table 1). The BgKL frequency differences between Shikoku and Osaka (P = 0.414), Shikoku and Chugoku (P = 0.137), and Osaka and Chugoku (P = 0.724) are not significant (Table 4). The BgKL frequency in Shikoku-Chugoku-Osaka (49.4%), however, is significantly higher than the nationwide average (P < 0.001).


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  		TABLE 4. Differences in the frequencies of BgKL variants: comparison of Shikoku, Osaka, and Chugokua

Of the isolates from Fukuoka, Kyushu Island, 31.3% were BgKL variants (Table 1). The BgKL frequency differences between Kyushu and Shikoku (P = 0.003), Kyushu and Shikoku-Chugoku (P = 0.006), and Kyushu and Shikoku-Osaka-Chugoku (P = 0.004) are significant, although the difference between Kyushu and Chugoku is not (P = 0.088) (Table 5).


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  		TABLE 5. Differences in the frequencies of BgKL variants: comparison of Kyushu and Okinawa with Shikoku, Chugoku, and Osakaa

None of the 14 isolates from Okinawa Island, 6 of which were from patients with genital HSV infections, was a BgKL variant (Table 1). The BgKL frequency difference between Okinawa and Kyushu (P = 0.020), Okinawa and Chugoku (P = 0.002), Okinawa and Shikoku (P < 0.001), and Okinawa and Shikoku-Osaka-Chugoku (P < 0.001) are all significant (Table 5).

Of the isolates from Shiga, 27.3% were BgKL variants, a lower frequency than in Osaka (Table 1). The BgKL frequency differences between Shiga and Shikoku (P = 0.010 by the Fisher exact test, P = 0.011 by chi-square test), Shiga and Shikoku-Osaka (P = 0.015 by the Fisher exact test, P = 0.017 by the Chi square test), Shiga and Shikoku-Chugoku (P = 0.030 by the Fisher exact test, P = 0.033 by the chi-square test), and Shiga and Shikoku-Osaka-Chugoku (P = 0.022 by the Fisher exact test, P = 0.031 by the chi-square test) are statistically significant (Table 6). The differences between Shiga and Osaka (P = 0.070) and Shiga and Chugoku (P = 0.097) are not (Table 6).


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  		TABLE 6. Differences in the frequencies of BgKL variants: comparison of Shiga with Shikoku, Chugoku, Osaka, Kyushu, and Okinawaa

Of the isolates from the Chubu region, 12.5% were BgKL variants; in particular, 15.0% in Aichi, 14.3% in Shizuoka, and 8.9% in Yamanashi (Table 1). The BgKL frequency in Chubu is slightly lower than in Shiga and statistically significantly lower than the nationwide average (P = 0.001). The BgKL frequency differences between Chubu and Osaka (P < 0.001), Chubu and Chugoku (P < 0.001), and Chubu and Shiga-Osaka (P < 0.001) are statistically significant, although the difference between Chubu and Shiga is not (P = 0.057) (Table 7). The pattern of significant differences in BgKL frequency between Aichi or Yamanashi and these areas is the same as that between Chubu and these areas (Table 7).


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  		TABLE 7. Differences in the frequencies of BgKL variants: comparison of Chubu, Aichi, and Yamanashi with Shiga, Osaka, Chugoku, Kyushu, and Okinawaa

Of the isolates from the Tohoku Region, 9.2% were BgKL variants; in particular, 5.9% in Fukushima, 6.7% in Miyagi, and 13.9% in Iwate (Table 1). The BgKL frequency in Tohoku is slightly lower than in Chubu. The BgKL frequency differences between Tohoku and Shiga (P = 0.017), Tohoku and Osaka-Shiga (P < 0.001), and Tohoku and Chugoku (P < 0.001) are statistically significant, but the differences between Tohoku and Chubu (P = 0.511) and Tohoku and Shiga-Aichi (P = 0.061) are not (Table 8).


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  		TABLE 8. Differences in the frequencies of BgKL variants: comparison of Tohoku and Chubu-Tohoku with Chubu, Shiga, Chugoku, Kyushu, and Okinawaa

The BgKL frequency in Chubu-Tohoku was significantly lower than the nationwide average (P < 0.001), and the BgKL frequency difference between Chubu-Tohoku and Shiga was statistically significant (P = 0.013) (Table 8). The BgKL frequency differences between Chubu-Tohoku and Shiga-Osaka (P < 0.001) and Chubu-Tohoku and Chugoku (P < 0.001) are also statistically significant (Table 8). We further compared the BgKL frequencies in Kyushu Island, west of Shikoku Island, and those in Osaka, Shiga, Chubu, and Tohoku, all east of Shikoku. The BgKL frequency difference between Kyushu and Shiga is not statistically significant (P = 0.827) (Table 6), but the differences between Kyushu and Osaka (P = 0.036), Kyushu and Chubu (P = 0.001) (Table 7), Kyushu and Tohoku (P < 0.001), and Kyushu and Chubu-Tohoku (P < 0.001) (Table 8) are all significant. These data indicate that the BgKL prevalence in Shiga is similar to that in Kyushu and that the BgKL frequencies in Chubu, Tohoku, and Chubu-Tohoku are lower than in Kyushu.

Similarly, the differences between Okinawa and Shiga (P = 0.042) (Table 6) and between Okinawa and Shikoku-Osaka-Chugoku (P < 0.001) (Table 5) are statistically significant, but the differences between Okinawa and Chubu (P = 0.225) (Table 7), Okinawa and Tohoku (P = 0.370) and Okinawa and Chubu-Tohoku (P = 0.371) (Table 8) are not. The results indicate that the BgKL frequency in Okinawa is similar to that in Chubu-Tohoku.

Taken together, these results indicate that the frequency of BgKL is high in the Shikoku (59.1%), Osaka (49.1%), and Chugoku (44.4%) (with a 49.4% average for the Shikoku-Osaka-Chugoku area), lower in Kyushu (31.3%) and in Shiga (27.3%), and lowest in Chubu (12.5%), Tohoku (9.2%), and Okinawa (0.0%), as depicted diagrammatically in Fig. 4.


Figure 4
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  		FIG. 4. Diagrammatic representation of the frequencies of HSV-1 BgKL variants in different geographic areas, indicating a geographic gradient distribution of the BgKL variant. The HSV-1 BgKL frequency level in geographic areas is indicated by black (high), gray (lower), and white (lowest) tones in accordance with the statistical significance of the frequency differences between adjacent areas. The P values for the geographic differences in the BgKL frequency are as follows: between Shikoku-Osaka-Chugoku and Kyushu (P = 0.004), between Kyushu and Okinawa (P = 0.020), between Shikoku-Osaka-Chugoku and Shiga (P = 0.022), and between Shiga and Chubu-Tohoku (P = 0.013). The broken line in the Chubu-Tohoku area indicates the location of Tokyo and the Kanto region, between the Chubu and Tohoku regions.

HSV-1 BgKL variant frequency in Tokyo. Isolates from the Tokyo metropolitan area, to which people from all over Japan have moved since it became the capital city four centuries ago, were included in this geographical study to see whether and how the demography of Tokyo has affected the BgKL frequency. The Tokyo BgKL frequency was 20.6% (Table 1), which is not significantly different from the average of the 15 other regions examined in the present study (28.3%) (P = 0.115), a finding consistent with the historical and recent demography of Tokyo.

Geographical distribution of multiple HSV-1 BgKL, SaCFJM, SaD/EL, SaGHM and KpMS variations. The frequency of HSV-1 clinical isolates with the BgKL variation and both the SaCFJM and the SaD/EL variations was 100% in Kyushu, Chugoku, and Shikoku; 95.2% in Osaka-Shiga; 91.7% isolates in Chubu-Tohoku; and 95.2% in Tokyo (Table 2). Thus, there are no significant differences in the frequency of HSV-1 isolates with these three RFLP variations in these regions.

The frequency of HSV-1 isolates with both the BgKL and the SaGHM variations was 92.9% in Shikoku, 93.3% in Chugoku, 90.5% in Osaka-Shiga, 90.9% in Kyushu, 75.0% in Chubu-Tohoku, and 90.5% in Tokyo (Table 2), indicating no statistically significant difference in these regions in the frequency of HSV-1 isolates with these two RFLP variations.

In addition, the frequency of HSV-1 isolates with both the KpMS and the BgKL variations was 100% in Kyushu, Chugoku, Shikoku, Osaka-Shiga, and Tokyo, and was 66.7% in Chubu-Tohoku (Table 3), indicating no statistically significant difference in these regions in the frequency of HSV-1 with the Bgl II and KpnI RFLP variations.

In summary, these results indicate that the frequency of the BgKL variant alone is a good measure of the frequency of the HSV-1 BgKL:SaCFJM:SaD/EL:SaGHM:KpMS variant in western, central and northeast Japan.


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DISCUSSION
 
The seroprevalence of HSV-1 is high in Japan (44) compared to among the Caucasian population in the United States (37, 38), similar to the situation with Epstein-Barr virus (6, 10) and human herpesvirus 6 (41). It is noteworthy that, during the 1960s and 1970s (11, 44), HSV-1 seroprevalence in Japan decreased to a level similar to that of the Caucasian population in the United States (37). By a combination of the high prevalence, transmission by intimate personal contact between HSV-1-excreting and susceptible individuals (37), and the lifelong latent infection-reactivation mode of HSV-1 (25, 37), HSV-1 isolates might have geographically variant characteristics if populations in different regions have been stable for a long time. HSV-1 viruses are occasionally reactivated and transmitted to new hosts. In this case, HSV-1 isolates from a given population should have accumulated diverse base substitutions by random mutation (4) since nonlethal HSV-1 mutants can survive because of latent infection for the life of the host. As expected from the accumulation of nonlethal mutations in HSV-1, variations in HSV-1 proteins (21, 27) and DNA restriction enzyme cleavage sites have been described (2, 3, 7, 26, 27) (14, 23, 28). RFLP analyses have documented multiple cleavage site changes in HSV-1 isolates (32, 33). This has raised the question of whether the clustering of cleavage site changes in geographically and racially distinct areas has been by host-dependent evolution of HSV-1 (32) or by the dispersion of conserved random mutations to different populations (25, 26). Recently, three HSV-1 genetic groups or genogroups have been identified on the basis of phylogenetic analyses of clinical HSV-1 isolates by Norberg et al. (20). In addition, it has been found that there are intergenic and intragenic recombinant viruses in clinical isolates, suggesting that most full-length HSV-1 genomes are a mosaic of segments from different genetic groups (20). Therefore, dispersion of HSV-1 between populations should play a role in the diversification of the HSV-1 genome. The high frequency of the HSV-1 BgKL variation enabled the studies reported here of large-scale analyses of the BgKL frequency in different geographic areas.

The major conclusions of these studies are as follows. First, the frequency of the BgKL variant in HSV-1 isolates is 27.0% in Japan, as determined from 636 HSV-1 isolates obtained from 16 different geographic areas. The length of the BglII K fragment in BgKL variants may be due to the loss of the BglII cleavage site between the K and Q fragments in the HSV-1 Justin strain DNA BglII cleavage map (14), although the Q fragment between K and O fragments has not been described in the F strain. This high frequency of the BgKL variant in Japan led us to detailed geographic and structural analyses of this RFLP variant.

Second, the striking picture of the geographic data on HSV-1 BgKL frequency is that there are two gradually decreasing gradient patterns, shown schematically in Fig. 4. One gradient is from Shikoku to the west, since the BgKL frequecy differences between Shikoku and Kyushu and between Kyushu and Okinawa are statistically significant. The other gradient is from Shikoku east to Shiga, Chubu, and Tohoku. The BgKL frequency difference is significant between Shikoku and Shiga and between Shiga and Tohoku. It has been reported (31) that the fraction of HSV-1 isolates with mutations at restriction enzyme cleavage sites, called HSV-1 "subtypes" in that report, in isolates from the Chugoku-Shikoku area was higher than in isolates from the northernmost island of Hokkaido. However, Japanese people only started to settle in Hokkaido Island about a century ago, late in the19th century, and this history may have affected the epidemiological data of Hokkaido. This report of large-scale analyses of HSV-1 isolates from many regions of Japan, where hundreds of generations of people have lived continuously, has shown that the frequency of the BgKL variants gradually decreases in a gradient from the Shikoku-Chugoku-Osaka region both to the west and to the east.

Third, 89.9% of the BgKL isolates have several additional RFLP variations that have been identified using SalI to detect the BgKL:SaCFJM, BgKL:SaD/EL, and BgKL:SaGHM variants. The SaCFJM variation indicates that the cleavage sites between both the SalI J and C fragments and the SalI F and J fragments have been lost. There is a SalI cleavage site in both the L component terminal repeat "b" sequence, in which the J and C fragments are separated by SalI, and the L internal inverted repeat "b'" sequence, in which the F and J fragments are separated; the base sequence of "b'" is identical to that of "b" except for its orientation (Fig. 2B) (14, 30). Therefore, The SaCFJM variation is interpretable by a single mutation in the "b" repeat sequence.

Furthermore, 96.3% of BgKL isolates have the KpnI cleavage pattern of the KpMS variant. It remains to be studied whether the BgKL variation is related to the genomic groups or recombinants of HSV-1 that have recently been described (20).

Fourth, the frequencies of HSV-1 BgKL:SaCFJM:SaD/EL:SaGHM:KpMS variants in BgKL variants was not different in HSV-1 clinical isolates from the eastern and western regions of Japan. Therefore, the extremely high frequency of BgKL isolates with these SalI and KpnI cleavage variations means that the BgKL frequency can be used as a measure of the frequency of HSV-1 variants with multiple mutations, at least at the BglII, SalI, and KpnI cleavage sites (i.e., the BgKL:SaCFJM:SaD/EL:SaGHM:KpMS variant) in Japan. Taking into consideration that the northern coast of Chugoku faces the Sea of Japan and the southern coast of Shikoku faces the Pacific Ocean, the bidirectional gradient of the BgKL:SaCFJM:SaD/EL:SaGHM:KpMS variant frequency suggests that a given HSV-1 variant can be geographically dispersed radially, unless the dispersion is blocked by a geographic barrier such as an ocean.

Fifth, it is possible that the geographical gradient of the HSV-1 BgKL:SaCFJM:SaD/EL:SaGHM:KpMS variant arose in multiple regions independently, but with different frequencies in different regions. This possibility seems unlikely. It was previously hypothesized that clustering of mutations at restriction endonuclease cleavage sites depends on geographically and racially distinct areas (32). However, the data reported here show such a big geographic difference in the frequency of the BgKL variant on the Honshu, Shikoku, Kyushu, and Okinawa islands, inhabited for hundreds of generations by an almost racially homogeneous people. Moreover, the geographic frequency gradients along the Japanese islands do not seem to be explicable in terms of the multiple independent origins of this variant. It is more likely that the BgKL:SaCFJM:SaD/EL:SaGHM:KpMS variant was dispersed from Shikoku Island to the surrounding regions and then to more distant regions. Consistent with this suggestion, Osaka has been the biggest center of commercial activity in western Japan for centuries. Shikoku Island also has held strong ties with the Chugoku region because they are only separated by the narrow Seto-naikai Inland sea and there are many small islands between the two regions. There was no statistical difference in BgKL frequency between the Tokyo metropolitan area and the 15 other regions examined in the present study, notwithstanding the geographic location of Tokyo in Eastern Honshu, supporting the suggestion above of BgKL dispersion. The BgKL dispersion suggested by these data and the historical and geographical conditions described here agree with the previously noted possibility that random mutations are conserved and dispersed in different populations, resulting in the clustering of RFLP variations (25, 26).

For millennia, the Japanese people have inhabited the Honshu, Shikoku, and Kyushu islands and have been relatively isolated from other nations. Immigration and commerce were prohibited and foreigners were not allowed to enter Japan from the 17th through the late 19th century. In addition, most Japanese lived in and around their birthplaces throughout their lives because of ancient social systems. These historical conditions maintained stable local communities until a century ago and may have played a role in the geographic distribution of the HSV-1 BgKL variant.

Finally, the findings presented here are the first epidemiologic data on the geographic dispersion of HSV-1. Why was the geographic dispersion of HSV-1 not reported previously? The major reason is the combination of the ubiquitous and high prevalence of HSV-1 in all populations examined in the world and the latency reactivation mode of infection of HSV-1 that lets HSV-1 mutants remain in the ganglions of hosts for their entire lives, escaping immunological surveillance. This allows more and more HSV-1 mutants to accumulate in every human population. The accumulation of such a great number of different HSV-1 mutants makes it hard to monitor the geographic dispersion of a particular HSV-1 mutant or variant, if it does not have a readily recognizable marker such as the BgKL marker. In addition, the slow speed of HSV-1 spread in a human population due to the mode of transmission by close physical contact between an infected person and a susceptible individual also hampers analyses of HSV-1 spread from one regional population to another. The gradient geographic profile of BgKL has been found in the present study because the proportion of the BgKL variant in HSV-1 isolates is high and because most of the HSV-1 isolates examined in this large-scale study were collected before the days of modern rapid and long-range mass transportation in Japan where local communities were geographically well maintained.

The conclusion based on the experimental results in the present study that an HSV-1 variant is dispersed geographically has implications for future studies on the epidemiology and diversification of HSV-1. There are probably many other distinct HSV-1 variants dispersing from different geographical regions to other regions in the world. Wide-ranging international dispersion of HSV-1 variants is now being accelerated due to a rapidly growing number of travelers, which will facilitate recombination between different variants. Thus, accelerated, wide diversification of HSV-1 may have an impact on the biological or pathological properties of HSV-1. Whether the BgKL variant has a virologic property that contributes to its geographic dispersion remains a subject for future study. Whether and how the BgKL variati is related to the recently reported HSV-1 genomic groups and the inter-genomic group recombinants (20) is also an intriguing question.

It has been shown in the present study that the frequency of the RFLP BgKL variant in clinical HSV-1 isolates reflects the BgKL:SaCFJM:SaD/EL:SaGHM:KpMS variant frequency and is a function of geography. The present study is the first to report differences in the prevalence of an HSV-1 virus containing multiple mutations between many regions where people of the same nation and race have lived for hundreds of generations. The results of the present study, that the BgKL frequency decreases in a gradient from Shikoku along the Japanese Islands, suggest that the BgKL variant was dispersed from Shikoku Island to neighboring regions then further afield. The implications of this investigation for future studies on HSV-1 are manifold.


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ACKNOWLEDGMENTS
 
The contributing members of the Cooperation Group for HSV-1 RFLP Variant Study in Japan are as follows: Seiichiro Hata, Osaka University Medical School, Suita City, Osaka Prefecture 565-0871; Hiroki Iga, University of Tokushima School of Dentistry, Tokushima City, Tokushima Prefecture 770-8504; 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 020-8505; Shunsaku Kobayashi, Yamaguchi University School of Medicine, Ube City, Yamaguchi Prefecture 755-8505; Yoshikatsu Ozaki, Shiga University of Medical Science, Shiga Prefecture 520-2192; 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; and Shigeru Yamamoto, Kurume University School of Medicine, Kurume City, Fukuoka Prefecture 830-0011.

We thank T. Funabashi, Toranomon Hospital, Tokyo, for HSV-1 clinical isolates from Tokyo, and 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; T. Ikushima, Shizuoka Institute of Environment and Hygiene, for HSV-1 clinical isolates from Shizuoka; 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; H. Yoshitake, School of Medicine, Ryukyu University, for HSV-1 clinical isolates from Okinawa; 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.

Financial support for this research was provided by grants-in-aid from the Yamanashi Institute of Health and the Ministry of Health and Welfare.


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FOOTNOTES
 
* Corresponding author. Mailing 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

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


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Journal of Clinical Microbiology, June 2006, p. 2109-2118, Vol. 44, No. 6
0095-1137/06/$08.00+0     doi:10.1128/JCM.02064-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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