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UV Light-Induced Reactivation of Herpes Simplex Virus Type 2 and Prevention by Acyclovir James F. Rooney, Stephen E. Straus, Margaret L. Mannix, Charles R. Wohlenberg, Steven Banks, Sanjay Jagannath, Jeffrey E. Brauer, and Abner Louis Notkins

Laboratory ofOral Medicine. National Institute ofDental Research. and Laboratory of Clinical Investigation. National Institute ofAllergy and Infectious Diseases. National Institutes ofHealth. Bethesda. Maryland

Many factors have been associated with reactivation of latent herpes simplex virus (HSV) infection [1-10]. Among them UV B light, a spectral component of sunlight, induces reactivation of HSV -I and -2 infections in patients with a history of cutaneous recurrences [11-14]. Previously we reported that UV light was capable of inducing HSV-2 recurrence at perigenital sites in 5 patients after 8 of 13 UV exposures [11]. This UV model system could prove useful in studying the pathogenesis of viral reactivation and for evaluating interventions to prevent recurrent disease. The present study was undertaken to examine UV -induced reactivation of HSV-2 in an expanded patient population and to determine whether acyclovir, an antiviral drug known to be effective in the treatment and suppression of spontaneous perigenital herpetic recurrences [15], would prevent UV light-induced viral infection.

Materials and Methods Study population. Otherwise healthy adults, aged 18-50 years, who had culture-confirmed HSV-2 recurrences at least four times per year involving nonoral and nongenital sites were

Received 28 January 1992; revised 28 April 1992. Presented in part: 30th Interscience Conference on Antimicrobial Agents and Chemotherapy. Atlanta, October 1990 (abstract 1108). Informed consent was obtained from all participants, and studies were conducted in compliance with the guidelines for clinical research of the National Institutes of Health and US Department of Health and Human Services. Reprints or correspondence: Dr. Abner Louis Notkins, Laboratory of Oral Medicine. National Institute of Dental Research, National Institutes of Health. Bldg. 30, Rm. 121, Bethesda, MD 20892. The Journal of Infectious Diseases This article is in the public domain.

1992;166:500-6

eligible for study. Patients were required to use contraception and must not have used antiviral agents within 30 days before study. Twenty-four patients were enrolled and had at least one UV study exposure. Twenty-one patients were female; 3 patients were black and 21 were Caucasian. The average age (±SE) was 38.0 ± 1.0 years. The average time with recurrent herpes infections was 8.3 ± 1.1 years, and the mean reported frequency of recurrent herpes was 8.7 ± 0.8 episodes/year. Patients reported a variety of sites of recurrent HSV infection including the genitals, buttocks, sacrum, abdomen, back, thigh, hand, and face. Although 12 patients reported a history ofgenital lesions, only 5 had had a recurrence in the previous year. UV light exposure. The sensitivity of skin to UV light was determined by establishing a minimum erythema dose (MED). A Psoralite model 2400 Hand and Foot Apparatus (Psoralite, Columbia, SC) was used; it emits light of primarily UV B wavelengths (295-320 nm), the part of the solar spectrum that is most potent in inducing cutaneous erythema. The light was centered 25.4 cm above the skin surface to yield a uniform light output at the skin surface of 1.0 mW/cm 2 • A cardboard template with appropriate apertures was placed over the skin site to be exposed. Remaining skin surfaces were fully draped. The MED was determined by exposing l-cm? areas of skin adjacent to the site of recurrent HSV infection to sequentially increasing doses of UV light [II, 16]. The test areas were observed at 24 h to grade the degree of erythema; the MED was defined as the minimum dose required to produce erythema with four sharp borders. The MED for patients varied from 20 to 240 s, being substantially longer for the 3 black patients (150.0 ± 52.0 s) than the Caucasians (36.2 ± 2.6 s). For patients who completed four UV study exposures, a second MED was ascertained at the exposure site to determine whether repeated exposure to UV light substantially alters the MED of such sites. Study design. Patients were exposed on one to four occasions (designated A, B, C, and D) to individualized 4 MED of

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UV B light is a potent stimulus for inducing reactivation oflatent herpes simplex virus (HSV) infections. Patients were enrolled in a double-blind placebo-controlled crossover trial to determine whether acyclovir can prevent UV light-induced HSV -2 recurrences. Twenty-four patients with a history of recurrent infection of perigenital sites (e.g., buttock, thigh) were exposed one to four times with 4 minimum erythema doses of UV light. Patients were given acyclovir 200 mg orally five times daily or matched placebo beginning 1 day before each exposure and continuing for 5 days after exposure. There were 13 UV-induced recurrences among 36 placebo treatments and 3 after 38 acyclovir treatments (P = .004). The mean time to recurrence (±SE) was 4.8 ± 0.3 days. HSV -2 lesions developed primarily at the site of UV exposure. The cutaneous distribution and timing of UV-induced recurrences was consistent with a neural localization (dorsal root ganglia) of latent viral infection. This UV light model permits direct examination of events leading to HSV-2 recurrences in humans and can be used to evaluate approaches to prevention.

1ID 1992; 166 (September)

UV-Induced HSV-2 Infection

analyze the site specificity of spontaneous versus UV-induced lesions. For paired analysis of acyclovir efficacy, the Cochran weighted mean difference was used [18]. Patients were included in this analysis if they received at least one course each of acyclovir and placebo and had at least one UV-induced site-specific HSV infection.

Results First UV light exposure. After the first exposure to 4 MED of UV light, 23 of 24 patients developed prominent erythema within 24 h. No blistering or significant discomfort was noted. Six site-specific recurrences (tables 1 and 2, figure 1A) developed within 7 days after UV exposure in 11 patients receiving placebo (rate of reactivation, 55%). In contrast, no site-specific recurrences were noted in 13 patients receiving acyclovir (table 2, P = .003). Subsequent UV light exposures. Subsequent UV exposures done at the precise site of previous UV exposure showed that the degree of erythema was markedly diminished compared with that from the first exposure. Rates of recurrence also were reduced (0/7 for placebo group, tables I and 2, nonoffset exposures). The reduction in erythema was attributed to tanning (hyperpigmentation and hyperkeratosis) induced by previous UV exposures. To quantitate the effect of UV exposure on responsiveness to subsequent exposures, a repeat MED was determined at the original exposure site within 60 days after the fourth (D) exposure for 14 patients. The average MED for 12 Caucasian patients increased from 32 to 102 s, while the average MED of2 black patients increased from 150 to 400 s. The increase in MED averaged 0.62 MED/exposure. On the basis of these observations, we offset the exposure site by 0.5 em for all subsequent exposures and used the same dose of UV light as used for first (A) exposures. This resulted in a 1.5- to 3.0-cm2 margin of brisk erythema (figure 1B) and an increase in rate of site-specific recurrence to 39% in the placebo group (table 2, offset exposures). Fifteen percent of the acyclovir-treated group also showed evidence of HSV infection. When all exposures (A-D) were compared, recurrence rates were significantly higher for placebo recipients than for acyclovir recipients (13/36 vs. 3/38, P = .004). A paired analysis compared the rate of site-specific events after UV exposures. Of the 24 patients, 11 received at least one course of acyclovir and one of placebo and had at least 1 site-specific recurrence, so could be evaluated in a paired analysis. The 3 patients who had recurrences while taking acyclovir were included in this analysis. The rate of recurrence per 100 days at risk after exposure was 9.2 for the placebo group and 2.6 for the acyclovir group (Cochran weighted mean difference, Z = 2.33, P = .02). Virology. Of 16 site-specific outbreaks, 13 were confirmed by viral culture and were determined by monoclonal

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UV light in a 3 X 3 ern area at a nongenital site of previous recurrence and were observed for documentation ofHSV recurrence. For each patient the site for UV light exposure was chosen on the basis of the frequency of recurrence and ease of administration of light to that location. The buttocks or sacrum was chosen as the UV exposure site for 18 patients. For the others, the thigh (3), abdomen or back (2), or hand (I) was used. Each exposure of a patient to UV light was separated from the previous exposure or previous HSV recurrence by ~ 3 weeks. For each study exposure, patients were instructed to take medication, either 200-mg acyclovir pills or indistinguishable placebo pills (provided by Burroughs Wellcome, Research Triangle Park, NC), five times daily for 6 days, beginning I day before exposure. Each patient was assigned to receive acyclovir for two exposures and placebo for two exposures, and the order of administration was randomized for each patient and was doubleblind. Patients were seen in clinic on the day ofexposure (day 0) and on days 3 and 5 after exposure or within 24 h ofonset ofany signs or symptoms of recurrence throughout the study. At these times symptoms were noted, lesions were examined, the location of lesions was noted, and specimens were taken for viral culture. Specimens for viral culture were also taken at the exposure site on days 0, 3, and 5 even in the absence of symptoms. Patients maintained records to document signs and symptoms of recurrences as well as medication use and compliance. They were followed for at least I year after the last UV exposure to document recurrences and any adverse effect of UV exposure. For the purpose of assessing antiviral efficacy, a site-specific UV light-induced reactivation was defined as a recurrence (documented by clinical symptoms, by positive viral culture, or both) within 7 days after exposure and located within I em of the margin of the burn. Other recurrences within 7 days ofexposure were considered to be non-site-specific, and recurrences at other times (i.e., >7 days) were considered to be spontaneous (also designated as site-specific or non-site-specific). When a recurrence was documented within 5 days of UV exposure, patients were advised to discontinue study medication and to take acyclovir (200-mg pills) five times daily for 5 days. Spontaneous recurrences could also be treated if desired. Viral studies. Specimens were collected and assayed for virus as previously described [II]. At least I isolate from each patient and all available positive cultures from UV -induced recurrences were typed by using a monoclonal antibody testing kit (Microtrak; Syva, Palo Alto, CA). All available isolates also were analyzed for sensitivity to acyclovir by using an 1251-radiolabeled HSV DNA probe for quantitating viral DNA (Hybriwix Herpes Antiviral Susceptibility Test Kit; Diagnostic Hybrids, Athens, OH) after incubation of virus isolates with various concentrations of acyclovir (0.1-4 JLg/mL). Results of duplicate testing were expressed as the ID 50 of acyclovir for each strain. Restriction endonuclease analysis was done as previously described [17], with four restriction enzymes: EcoRI, Kpnl, HindIII, and BamHI. Profiles on agarose gel electrophoresis were compared with those of standard laboratory strains of HSV-2. Statistical methods. Fisher's exact test was used to analyze the effect of acyclovir treatment on HSV recurrence, to examine the effect of offset UV exposures on viral recurrence, and to

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Table 1. Treatment assignments and HSV recurrences after UV exposures.

Time (days) to recurrence

Time (days) to recurrence

Non-sitespecific

Site-specific

Site-specific

Course* Patient no.

Acyclovir

Course*

Placebo

Days

Days

Days

Days

0-7

8-30

0-7

8-30

A Dt A

Patient no. 12 13 14

Bt ct 2

Acyclovir

4 5

5

3

29

17

5 13 4

27

B C 17

D A

5 5 13

B C D 18

B C D A Bt

5 3

C D A

3 19 20 21

17

5

A B

13

12 II

C D

3

A

6

A

5 5

17

A A

3

B 20

Bt C

22

B

3

C

17

D

D A

5

14

A

23

Bt

B C

II

10

A

16

5

D A

10

4

C D

ct

9

A

D A B

15

Bt A

8

8-30

A

Bt 7

Days

0-7

B

A A

6

Days

8-30

C

7

A Bt ct D

Days

0-7

C

6

D A

24

Bt

5

C

I

D

25 21

D A

26

B C

4

*A-D, first through fourth course. t Subsequent nonotfset exposures. t Results were discounted from final analysis because ofaccidental UV exposure outside of target site.

antibody stammg to be HSV-2. Restriction endonuclease analysis on 10 UV-induced virus isolates from 7 patients demonstrated that they were identical to the virus strains that the respective patient shed during a previous spontaneous recurrence (data not shown). Specimens taken for culture from the exposure site in the absence of clinical lesions (on days 0, 3, and 5 after UV exposure) were negative except for

a specimen from patient no. 16, who shed virus asymptomatically on day 5 after UV exposure while taking acyclovir. Acyclovir sensitivities of29 virus isolates from 21 patients were determined, including isolates from the 3 UV-induced recurrences that developed in patients taking acyclovir. All isolates were sensitive to acyclovir (ID 50 < I JLg/mL). Time to recurrence and site specificity ofrecurrences. The

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C

Days Placebo

A

Btt 3

Non-sitespecific

UY-Induced HSY-2 Infection

JID 1992; 166 (September)

Table 2. Site-specific HSV recurrences within 7 days after UV exposure: effect of acyclovir. Treatment group

First exposure Subsequent exposures Nonoffset burn Offset burn Combined results First and offset exposures All exposures

Placebo

Acyclovir

p*

6/11 (55)

0/13

.003

0/7 7/18(39)

0/5 3/20 (15)

13/29(45) 13/36 (36)

3/33 (9) 3/38 (8)

>.5 .14 .003 .004

gesting that UV-induced recurrences are the result of regional pathways capable of modulating viral latency and reactivation. To determine whether the anatomic distribution of recurrences (site-specific vs. non-site-specific) after UV exposure varied from those of spontaneous lesions, the location of the recurrences within 7 days after UV exposure were compared with the location of spontaneous recurrences (i.e., after day 7). Only spontaneous recurrences that were observed in the clinic were evaluated. Ten ( 15%) of 65 documented spontaneous recurrences were site-specific, whereas 16 (67%) of 24 recurrences documented in the week after UV exposures were site-specific (P < .001, two-tailed Fisher's exact test).

Discussion mean time (±SE) to site-specific recurrence in the first week after UV exposure was 4.8 ± 0.3 days for placebo and 5.0 ± 0.0 days for acyclovir recipients (figure 2). No increase in HSV outbreaks was noted after discontinuation of acyclovir on day 5 (figure 2), suggesting that the stimuli to reactivation following UV-light administration persists for

UV light-induced reactivation of herpes simplex virus type 2 and prevention by acyclovir.

UV B light is a potent stimulus for inducing reactivation of latent herpes simplex virus (HSV) infections. Patients were enrolled in a double-blind pl...
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