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Short Communication |
1 Herpesvirus Laboratory, Department of Virology I, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
2 BML General Institute, Matoba 1361-1, Kawagoe, Saitama 350-1101, Japan
3 Department of Dermatology, Doai Kinen Hospital (Fraternity Memorial Hospital, Japan), Yokoami 2-1-11 Sumida-ku, Tokyo 130-8587, Japan
4 Yamanashi Institute of Health, Kofu City, Yamanashi Prefecture, Japan
5 Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030-3498, USA
Correspondence
Kazuo Yanagi
kyanagi{at}nih.go.jp
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Present address: St Louis Laboratories, Pfizer Inc., 700 Chesterfield Parkway West, Mail Zone AA3C Chesterfield, MO 63017, USA. 
Present address: AIDS Research Center National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku, Tokyo 162-8640, Japan.
Two supplementary figures and data supplements A–K are available with the online version of this paper.
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, 1989a) caused by loss of the BglII cleavage site between the BglII K and Q/13 fragments (Eda et al., 2007). BgKL is a good surrogate marker for the HSV-1 multiple mutant BgKL:SaCFJM:SaEL:SaGHM:KpMS, where SaCFJM denotes loss of SalI fragments C, F and J, SaEL denotes a larger SalI L fragment, SaGHM denotes loss of SalI G and H fragments and KpMS denotes a smaller KpnI M fragment (Eda et al., 2007; Ozawa et al., 2006). The prevalence of BgKL in HSV-1 clinical isolates, referred to as its relative frequency (RF), is 27 % in Japan (Ozawa et al., 2006). Another HSV-1 RFLP variant BgOL (Ozawa et al., 1989b) has lost the BglII cleavage site between the BglII Q/13 and O fragments (Eda et al., 2007). It has been proposed that BgKL spread and replaced BgOL in Japan, based on the geographical distribution profiles of BgKL and BgOL (Eda et al., 2007; Ozawa et al., 1989b, 2006</xref>) and the high RF of BgKL in orolabial infections (Ozawa et al., 2007).
In the present study, we compared the pathogenic and epidemiological properties of HSV-1 BgKL, BgOL and non-BgKL:non-BgOL variants in mice and sumo wrestlers with herpes gladiatorum (HG). Central nervous system (CNS) destruction is a major consequence of experimental murine disease induced by HSV-1 (Dix et al., 1983b; Goel et al., 2002; Mao & Rosenthal, 2003; Richards et al., 1981; Roizman & Knipe, 2001). HG has been documented among wrestlers and rugby players (Anderson, 2003; Becker, 1992; Belongia et al., 1991; De Bernardo, 1992; Turbeville et al., 2006; White & Grant-Kels, 1984). HSV-1 isolates from HG cases in professional sumo wrestlers that were previously described by Asano et al. (1994) were analysed further because the high HG frequency in these wrestlers and their traditional lifestyle, in which they live together in communal and isolated sumo stables, facilitated studies of HSV-1 epidemiology.
For pathogenicity experiments in mice, the HSV-1 isolates (and their isolation sites) used were: BgKL isolates RM48 (skin), TS9 (gingivostomatitis) and KH424 (keratitis); BgOL isolates RM57 (mouth), Y82-469 (mouth) and IW30 (lips); and non-BgKL:non-BgOL isolates RK [Kaposi's varicelliform eruption (KVE)], TS5 (lips) and TS6 (gingivostomatitis). The three non-BgKL:non-BgOL isolates showed the same RFLP profiles as strain F (kindly provided by Dr B. Roizman, University of Chicago) with BglII, SalI, KpnI, BamHI, HindIII, HpaI and EcoRI (Ozawa et al., 2006). The pathogenicity of HSV-1 clinical isolates was assayed (Beers et al., 1993, 1995) by inoculating BALB/c mice with a dilution series of each virus. Each mouse was injected with a 20 µl virus suspension for intracranial inoculations and a 100 µl virus suspension for intraperitoneal inoculations. Five mice were injected for each HSV-1 suspension dilution.
Neuroinvasiveness of the clincial isolates in mice was examined by intraperitoneal inoculation (Supplementary Fig. S1a, available in JGV Online) and indicated that the log10 LD50 values were: >6.0 (KH424), 3.2 (RM48) and 4.0 (TS9) for BgKL (i.e. a >2.8 log LD50 range); 2.4 (RM57), 4.0 (IW30) and 2.6 (Y82-469) for BgOL (i.e. a 1.6 log LD50 range); and 2.6 (RK), 3.0 (TS6) and >6.0 (TS5) for non-BgKL:non-BgOL, (i.e. a >3 log LD50 range). The neurovirulence of the clincial isolates was examined by intracranial inoculation in mice (Supplementary Fig. S1b) and indicated that the log10 LD50 values were: <1.0 (KH424), <1.0 (RM48) and 1.8 (TS9) for BgKL; <1.0 (RM57), 1.4 (IW30) and <1.0 (Y82-469) for BgOL; and <1.0 (RK), <1.0 (TS6) and 3.8 (TS5) for non-BgKL:non-BgOL (i.e. similar log10 LD50 values for isolates of the three variants). These results indicate that the pathogenicity of BgKL isolates in mice, measured as LD50 values, varied widely between isolates and was similar to that for BgOL and non-BgKL:non-BgOL isolates and for HSV-1 clinical isolates (Bergstrom et al., 1990; Dix et al., 1983a, 1983b; Mao & Rosenthal, 2003; Richards et al., 1981) and mutants (Roizman & Knipe, 2001).
Clinical specimens and HSV-1 isolates from sumo wrestlers, described previously by Asano et al. (1994), were taken between August 1989 and July 1994 from young professional sumo wrestlers with HG living in eight different sumo stables in Tokyo: Tomozuna, Takasago, Oguruma, Nishonoseki, Tatsunami, Kokonoe, Dewanoumi and Wakamatsu (designated stables B, D, E, G, I, J, K and L, respectively). Clinical diagnosis was at the Doai Kinen Hospital (The Fraternity Memorial Hospital). One wrestler in stable D developed headaches and died 2 months after clinical diagnosis of HG in 1989 and one wrestler in stable F was diagnosed with herpes encephalitis in 1990; specimens were not obtained from these two wrestlers, however, this indicates that HSV-1 infections can be fatal in some cases and as such they warrant investigation. Specimens were inoculated into MRC-5 (ATCC, CCL-171) cell cultures. Thirty-nine sumo wrestlers were diagnosed with HG; HSV-1 was isolated from 22 of these wrestlers (Asano et al., 1994) and HSV-2 was isolated from another wrestler (data not shown). Virus isolates were propagated on Vero cell (Yasumura & Kawakita, 1988) monolayers and their HSV type was identified using HSV type-specific monoclonal antibodies, with plaque titration performed as previously described (Yanagi, 1981). The virus suspensions used in this study were prepared from virus stocks that had not been passaged more than five times after isolation. Neutralizing and complement-requiring neutralizing (CRN) antibodies against HSV-1 in serum specimens were titrated soon after the specimens were taken, as previously described (Asano et al., 1994; Yoshino & Abe, 1981; Yoshino & Isono, 1978). Complement (0.25 units) was added to each well of 96-well micro-test plates for CRN antibody titrations (Yoshino & Abe, 1981). RFLP analyses were performed as previously described (Eda et al., 2007; Ozawa et al., 2006) using the restriction endonuclease maps of strain F as references (Hayward et al., 1975; Locker & Frenkel, 1979; Roizman, 1979; Roizman & Tognon, 1983). The terminal internal and ‘joint’ sequences containing the 280 bp repeat, consisting predominantly of the terminal reiterated sequence ‘a’ (Davison & Wilkie, 1981; Locker & Frenkel, 1979; Roizman, 1979) (Fig. 1), were excluded from the RFLP analyses (Locker & Frenkel, 1979; Roizman & Tognon, 1983; Wagner & Summers, 1978). Several subterminal fragments with size variability, as previously described by Roizman & Tognon (1983), are indicated in Fig. 1.
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; Yanagi, 1981; Yoshino et al., 1977; Yoshino & Isono, 1978) appears in early immune sera of infected patients and experimental animals, as described previously (Yoshino & Isono, 1978; Yoshino & Taniguchi, 1966; Zheng & Hsiung, 1984), and CRN antibody titres are higher than neutralizing antibody titres in early sera (Yoshino & Isono, 1978; Yoshino & Taniguchi, 1966).
Five sumo wrestler HG cases in stable L presented almost concurrently (Table 1). One HG case (HG37IY) presented 2 weeks later, another (HG38OH) presented 1 month after HG37IY and another (HG39OY) presented 8 months after HG38OH (Table 1). Sera from five HG cases obtained before or at the onset of HG were negative (i.e. titre <4) for HSV-1-neutralizing and CRN antibodies. Four of these cases seroconverted, as determined by their HSV-1-neutralizing and CRN antibody titres, but paired sera were not obtained for the fifth case (HG35HH) (Table 1). These data indicated that these five HG cases were primary HSV-1 infections. In three other cases from stable L, the HSV-1-neutralizing and CRN antibodies were positive before or at HG onset/diagnosis (Table 1), indicating that these cases were not primary HSV-1 infections. In four cases from stable E (Table 1), HSV-1-neutralizing and CRN antibodies were negative in two cases (HG24MT and HG25SM) and positive in two cases (HG15HK and HG16AT) at the time of HG diagnosis, indicating that the former cases were primary HSV-1 infections and the latter were not. HG recurred in a number of sumo wrestlers, as described in data supplement A.
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32 and CRN antibody titres 128 (Table 1
). These neutralizing antibody levels were higher than those in sera from wrestlers with HG taken before or at the onset of HG (Table 1).
Five different HSV-1 isolates with distinct RFLP profiles, namely different HSV-1 strains, were involved in HG cases in stable L and two were involved in HG cases in stable D. In all other stables, one HSV-1 strain was responsible for all HG cases in that stable (Table 2 and data supplements B–G).
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). These BgKL strains were all the BgKL:SaCFJM:SaEL:SaGHM:KpMS variant except for BgKL(D3). The number of stables in which BgKL was isolated (six) was larger than that in which non-BgKL was isolated (three) (Table 2). Furthermore, for sumo wrestlers with HG, the BgKL RF value of 81 % (17 of the 21 BgKL-infected wrestlers with skin or eyelids lesions; Table 2) was significantly higher (data supplement H) than the BgKL RF of 27 % in the general population in Japan (Ozawa et al., 2007).
In seven of the 22 (32 %) HG cases, eczematous lesions with systemic illness were observed and these cases were diagnosed as KVE (also called eczema herpeticum) (Kramer et al., 2004; Marcus et al., 2005; Ruchman, 1954) (Table 2). All HSV-1 isolates from these KVE cases were BgKL (Table 2).
Concurrent infections of different HSV-1 strains in HG cases has been suggested previously (Belongia et al., 1991; Dworkin et al., 1999) but the RFLP data were ambiguous. The present study is, to our knowledge, the first to report precise RFLP analyses, with many restriction endonucleases, of HSV-1 isolates from HG cases. The living conditions of professional sumo wrestlers suggested that the source of primary HSV-1 infections among sumo wrestlers in each stable was their fellow wrestlers. Three different BgKL strains and two different non-BgKL:non-BgOL strains were isolated from eight HG cases in stable L wrestlers. Five of these cases were primary HSV-1 infections. These results indicate that concurrent HSV-1 infections caused by different HSV-1 strains in wrestlers within the same sumo stable are common (data supplement J).
In the field of sports medicine, recurrent HG has usually been diagnosed simply based on symptoms and the wrestler's medical history (Anderson, 2005; Becker et al., 1988; Strauss et al., 1989; Turbeville et al., 2006). Sumo wrestlers' HG cases were caused by non-primary HSV-1 infections as well as by primary HSV-1 infections. Repeated HG recurrences within a short period of time and isolation of HSV-1 isolates with different RFLP patterns from the same sumo stable suggested reactivation of latent HSV-1 infections in these wrestlers. The frequent severe skin damage of sumo wrestlers may impair subcutaneous nerve cells and the strong physiological and mental stress resulting from their hard practice regime may affect their immunological status (data supplement I). This may explain why HG recurred in a number of sumo wresters. The possibility of an immunological effect is supported by the results reported here, since wrestlers who did not develop HG had high titres of neutralizing antibodies compared with pre-HG sera from wrestlers who were susceptible to recurrent HG.
The high association of BgKL with sumo wrestlers' KVE is statistically significant (P=0.03, Fisher's exact test). Therefore, a BgKL-related mutation(s) may enhance HSV-1 pathogenicity in skin or affect the host immunological response.
Finally, the BgKL RF in professional sumo wrestler HG cases was higher than that in the general population in Japan; this difference was statistically significant. In addition, the number of different BgKL strains isolated from sumo wrestler HG cases was larger than the number of BgOL and non-BgKL:non-BgOL variants isolated from these cases; the number of stables in which BgKL was isolated was also larger than that in which non-BgKL was isolated.
Taken together, these results suggest that latent BgKL infections were reactivated and transmitted more efficiently than latent non-BgKL infections in these cases (data supplement K). Future studies in this laboratory will analyse the genome sequences of these BgKL clinical isolates. As far as we are aware, the present study is the first to suggest that the efficiency of HSV-1 reactivation/recurrence in humans may depend on the HSV-1 variant or strain and it supports the BgKL dispersion hypothesis (Eda et al., 2007; Ozawa et al., 2006, 2007).
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ACKNOWLEDGEMENTS |
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Received 21 April 2008;
accepted 10 June 2008.
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