Method of inhibiting formation of infectious herpes virus particles

The present invention provides a method of inhibiting the formation of infectious herpes virus particles, particularly infectious herpes simplex virus (HSV) particles, in a host cell. The method involves administering an effective amount of a hydroxylated stilbene, particularly resveratrol, to a herpes virus infected host cell. The present invention also provides a method of treating a herpes virus infection, particularly an HSV infection. The method comprises administering a topical composition comprising a therapeutically effective amount of a hydroxylated stilbene to a herpes virus-infected site. The present invention also relates to a topical composition for treating a herpes virus infection selected from the group consisting of an HSV infection, a cytomegalovirus infection, and a varicella zoster virus infection. The present invention also provides a method of reducing the cytopathic effect of HSV on mammalian cells. The method involves administering resveratrol to the host cell, either in vitro or in vivo, in an amount sufficient to inhibit replication of HSV-1 or HSV-2 within the host cell.

BACKGROUND
 Human herpes viruses can infect host cells in virtually any organ of the
 human body. Replication of a herpes virus within an infected host cell
 leads to lysis of the infected cell and the release of large numbers of
 infectious virus. The infectious particles released from the lysed cell
 can infect and destroy other cells at or near the site of the initial
 infection. These infectious particles can also be transmitted to a
 non-infected individual. These infectious particles can also enter and
 remain latent, i.e., in the non-replicative state, in other cells of the
 afflicted individual for life. This life-long infection serves as a
 reservoir of infectious virus for recurrent infections in the afflicted
 individual and as a source of infection for an unwitting contact.
 At least four of the human herpes viruses, including herpes simplex virus
 type 1 (HSV- 1), herpes simplex virus type 2 (HSV-2), cytomegalovirus
 (CMV), and varicella zoster virus (VZV) are known to infect and cause
 lesions in the eye of certain infected individuals. Together, these four
 viruses are the leading cause of infectious blindness in the developed
 world.
 HSV-1 primarily infects the oral cavity, while HSV-2 primarily infects
 genital sites. However, any area of the body, including the eye, skin and
 brain, can be infected with either type of HSV. Generally, HSV is
 transmitted to a non-infected individual by direct contact with the
 infected site of the infected individual.
 The initial symptoms of a primary or recurrent HSV infection include
 tingling, pain, and/or parasthesia at the site of infection. This is
 followed by formation of a lesion at the infected site, i.e., in the oral
 cavity, eye, skin, or reproductive tract. Healing typically occurs in
 approximately ten to fourteen days.
 The immune reaction that occurs in response to an HSV infection prevents
 dissemination of the virus thtoughout the body of the immmunocompetent
 individual. Such immune reaction. however, does not eliminate all
 infectious HSV particles from the body of the afflicted individual. The
 virus particles that are not killed by the immune response move along the
 nerve path to the ganglia of the infected individual where they remain in
 a state of latency. In response to a variety of stimuli including stress,
 environmental factors, other medications, food additives or food
 substances, the infectious virus particles may leave the ganglia and cause
 a recurrent infection at or near the original site of infection. In those
 HSV-infected individuals who are immunosuppressed or who lack a
 well-developed immune system, such as neonates, dissemination of the virus
 particles from the infected site can also occur and lead to
 life-threatening complications, including encephalitis.
 VZV, which is transmitted by the respiratory route is the cause of
 chickenpox, a disease which is characterized by a maculopapular rash on
 the skin of the infected individual. As the clinical infection resolves,
 the virus enters a state of latency in the ganglia, only to reoccur in
 some individuals as herpes zoster or "shingles" The reoccurring skin
 lesions remain closely associated with the dermatome, causing intense pain
 and itching in the afflicted individual.
 CMV is more ubiquitous and may be transmitted in bodily fluids. The exact
 site of latency of CMV has not been precisely identified, but is thought
 to be leukocytes of the infected host. Although CMV does not cause
 vesicular lesions, it does cause a rash.
 There are no known cures for infections with human herpes viruses, i.e.,
 methods of eliminating the virus from the body of the infected individual.
 In addition, there are very few methods for blocking the formation of
 infectious herpes virus particles and thereby reducing the frequency,
 severity, or duration of a herpes virus-induced infection and the
 likelihood of recurrence of infection in the latently-infected individual.
 Thus, it is desirable to have additional methods for inhibiting the
 formation of infectious herpes virus particles. Such method is useful for
 limiting the severity of a herpes virus infection within an infected
 individual and the likelihood of transmission of the herpes virus
 infection from the infected individual to a non-infected individual.
 SUMMARY OF THE INVENTION
 The present invention provides a new method of inhibiting the formation of
 infectious herpes virus particles, particularly infectious HSV particles,
 in a host cell. The method involves administering a hydroxylated stilbene,
 particularly resveratrol, to a herpes virus infected host cell. The
 hydroxylated stilbene is administered to the host cell in an amount
 sufficient to inhibit replication of the virus in the virus-infected host
 cell. Such method is useful for reducing the cytopathic effect of a herpes
 virus infection. Such method is also useful for preventing the spread of
 the herpes virus from a virus-infected host cell to a non-infected host
 cell. Such method is also useful for establishing a model system for
 studying the molecular events that occur during replication of herpes
 virus and for studying the factors that trigger replication of a latent
 herpes virus, particularly replication of latent HSV.
 The present invention also provides a method of treating a herpes virus
 infection, particularly an HSV infection. The method comprises
 administering a topical composition comprising a therapeutically effective
 amount of a hydroxylated stilbene, particularly resveratrol, to a herpes
 virus-infected site. The present invention also relates to a topical
 composition for treating a herpes virus infection selected from the group
 consisting of an HSV infection, a CMV infection, and a VZV infection.
 The present invention also provides a method of reducing the cytopathic
 effect of HSV on mammalian cells. The method involves administering
 resveratrol to the host cell, either in vitro or in vivo, in an amount
 sufficient to inhibit replication of HSV-1 or HSV-2 within a
 virus-infected host cell.

DETAILED DESCRIPTION OF THE INVENTION
 In one aspect, the present invention provides a method of inhibiting
 formation of infectious herpes virus particles, particularly infectious
 HSV particles, in a host cell. The method comprises administering a
 hydroxylated stilbene to the host cell. The hydoxylated stilbene is
 administered in an amount sufficient to or effective to inhibit
 replication of the herpes virus within the infected cell. Preferably, the
 hydroxylated stilbene, is administered to the host cell either prior to
 infection of the host cell with the virus or preferably, within six hours
 after infection of the host cell with the virus.
 Preferably, the hydroxylated stilbene is administered to the host cell by
 contacting the host cell with or exposing the host cell to a composition
 comprising the hydroxylated stilbene. For example, in vitro, the method
 comprises adding a hydroxylated stilbene to the culture medium of herpes
 virus-infected host cells. In the case of cultured cells, the hydroxylated
 stilbene is added to the medium, preferably before the host cells are
 infected with the virus or within six hours after the host cells are
 infected with the virus. In the case of ganglia, which serves as an organ
 culture model system for studying latency of herpes viruses, particularly
 for studying latency of HSV, the hydroxylated stilbene is added to the
 medium after the ganglia are excised from the latently-infected host. With
 respect to HSV, good results have been obtained by exposing cultured host
 cells or HSV-infected ganglia to the hydroxylated stilbene, resveratrol,
 at a concentration which is greater than 10 .mu.g/ml and less than 100
 .mu.g/ml of culture medium.
 It has been determined that treatment of cultured cells in accordance with
 the present method is non-toxic to cells and blocks replication of HSV at
 some early stage in the replicative cycle of this human herpes virus. It
 has also been determined that the effect of resveratrol on HSV replication
 is reversible. Typical of the herpes viruses, HSV replication occurs in
 phases, with each phase being dependent on the successful completion of
 the prior phase. The "immediate early phase" occurs at 1-3 hours after
 infection and is associated with regulatory and synthetic events. The
 "early phase" occurs 3-6 hours after infection and is also associated with
 regulatory and synthetic events, particularly the synthesis of virus DNA.
 The "late phase" occurs 6-10 hours after infection and is associated with
 final synthetic events and assembly of viral components into infections
 virions. Accordingly, since all herpes viruses have in common a
 replicative scheme that progresses through similar and distinct phases,
 such method is useful for establishing model systems for studying the
 molecular events that occur during replication of all herpes viruses. For
 example, mammalian cell cultures incubated in the presence and absence of
 resveratrol may be used to identify cellular factors that are involved in
 regulating herpes virus synthetic events. Such cell cultures may also be
 employed to characterize the role of HSV gene products in the replication
 of infectious virus, particularly those proteins and factors whose
 function are currently unknown.
 Such method is also useftil for establishing a model system for studying
 latency of herpes viruses, particularly latency of the herpes viruses that
 remain latent in the ganglia, such as for example HSV and VZV. Such model
 system is useful for characterizing the extracellular factors such as for
 example hormones and cytokines, as well as the intracellular factors and
 molecular events that trigger replication of latent herpes viruses.
 In another aspect, the method comprises administering a pharmaceutical
 composition, preferably a topical composition, comprising a
 therapeutically effective amount of a hydroxylated stilbene, preferably
 resveratrol, to the site of the infection. As used herein "site of the
 infection" means a previously uninfected site which is expected to come
 into contact with a herpes virus-infected site or the site of a current or
 prior herpes virus-induced lesion. Such method is particularly useful for
 treating local herpes virus infections, such as for example, HSV-induced
 skin lesions, HSV-induced eye infections, HSV-induced lesions of the
 reproductive tract, CMV-induced eye lesions, and VZV-induced eye lesions.
 In such cases, it is preferred that the hydroxylated stilbene, more
 preferably resveratrol, be applied directly to the infected site. It is
 preferred that the hydroxylated stilbene be administered to the
 herpesvirus-infected site in the form of an aqueous solution or in the
 form of a salve. For eye infections, it is preferred that an aqueous
 solution of the hydroxylated stilbene, more preferably resveratrol, be
 administered as an eye drop. For herpesvirus skin lesions, such as for
 example, HSV-induced skin lesions, or HSV-induced lesions of the
 reproductive tract, it is preferred that the composition be applied
 topically.
 HYDROXYLATED STILBENES
 The structural skeleton, of the compound employed in the present invention,
 i.e., the hydroxylated stilbene, comprises two aromatic rings joined by a
 methylene bridge. Preferably, the hydroxylated stilbene is a
 polyhydroxylated stilbene, more preferably a trihydroxystilbene or a
 tetrahydroxystilbene, most preferably the tri-hydroxylated stilbene,
 3,5,4'-trihdyroxystilbene, also known as resveratrol, or a derivative
 thereof. Resveratrol in either the cis form or trans form is suitable.
 Derivatives of resveratrol as used herein refers to compounds in which one
 or two of the hydroxyl functions of resveratrol are replaced with other
 moieties such as, for example, pterostilbene in which the hydroxyl
 functions at positions 3 and 5 on the disubstituted aromatic ring are
 methoxylated and the .beta.-glucosidc derivative polydatin or piceid, in
 which one of the hydroxyl functions on the disubstituted aromatic ring is
 replaced with glucose; as well as polymers of the parent compound
 resveratrol. Such polymers have been given the name viniferins. Methods
 for producing the hydroxylated stilbenes are described in Moreana-Manas,
 M. et al, Anal Quim (1985) 81:157-161; Jeandet, P. et al, Am J. Enol Vitic
 (1991) 42:41-6; Goldberg DM et al. Anal Chem (1994) 66:3959-63, Murakami,
 S et al, Biochem Pharmacol. (1992) 44:1947-51; and Thakkar, K et al, J.
 Med Chem (1993) 36:2650-51, which are incorporated herein by reference.
 Resveratrol and 3,3',4,5'-tetrahydroxy-trans-stilbene, known as
 piceatoannol, are also available commercially from Sigma Chemical Co., St.
 Louis, Mo.
 TOPICAL COMPOSITION
 A pharmaceutical composition comprising a therapeutically effective amount
 of a hydroxylated stilbene, preferably a polyhydroxylated stilbene, more
 preferably rcsveratrol or a derivative thereof, and a pharmaceutically
 acceptable carrier, preferably a topical carrier is administered to or
 proximate to the cells known to be infected with, or suspected of being
 infected with the virus, or expected to come into contact with infectious
 virus. Preferably, the composition comprises a relatively inert topical
 carrier. Many such carriers are routinely used and can be identified by
 reference to pharmaceutical texts. Examples include polyethylene glycols,
 polypropylene copolymers, and some water soluble gels. Such a composition,
 referred to hereinafter as the "topical composition", may also contain
 diluents, fillers, salts, buffers, stabilizers, solubilizers, and other
 pharmaceutically acceptable materials well known in the art. The term
 "pharmaceutically acceptable" means a non-toxic material that does not
 interfere with the effectiveness of the antiviral activity of the
 hydroxylated stilbene.
 In practicing the present method of treatment or use, a pnarmaceutical
 composition comprising a therapeutically effective amount of the
 hydroxylated stilbene, preferably resveratrol, is applied to the site of
 infection in the host subject before or after the host subject is exposed
 to the virus. Such composition is particularly effective in treating
 infections of the eye, oral cavity and vagina as well as border areas of
 the lips and rectumn. In the case of oral administration, dentrifices,
 mouthwashes, tooth paste or gels, or mouth sprays are used. Vaginal or
 rectal administration may be by the usual carriers such as douches, foams,
 creams, ointments, jellies, and suppositories, the longer lasting forms
 being preferred. Ocular administration is preferably by ophthalmic
 ointments or solutions. Lip treatment is, preferably, in the form of a
 gel.
 The topical composition may further contain other agents which either
 enhance the activity of the hydroxylated stilbene or complement its
 activity or use in treating the viral disease. Such additional factors
 and/or agents may be included in the pharmaceutical composition to produce
 a synergistic effect with the hydroxylated stilbene, or to minimize side
 effects. The topical composition may also contain an agent which enhances
 uptake of the hydroxylated stilbene.
 Preferably the topical composition comprises a solvent for the hydroxylated
 stilbene, such as, for example, an alcohol. A liquid carrier such as
 water, petroleum, oils of animal or plant origin such as peanut oil,
 mineral oil, soybean oil, or sesame oil, corn oil, or synthetic oils may
 be added. The liquid form of the pharmaceutical composition may further
 contain a physiological saline solution, dextrose or other saccharide
 solution, or glycols such as ethylene glycol, propylene glycol or
 polyethylene glycol. The preparation of such topical composition having
 suitable pH, isotonicity, and stability, is within the skill in the art.
 The topical composition of the invention may be in the form of a liposome
 in which the hydroxylated stilbene is combined with amphipathic agents
 such as lipids which exist in aggregated form as micelles, insoluble
 monolayers, liquid crystals, or lamellar layers in aqueous solution.
 Suitable lipids for liposomal formulation include, without limitation,
 monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids,
 saponin, bile acids, and the like. Preparation of such liposomal
 formulations is within the level of skill in the art.
 DOSAGE
 The hydroxylated stilbene, preferably a polyhydroxylate stilbene, more
 preferably resveratrol or a derivative thereof is administered to the site
 of infection in the host subject in a therapeutically effective amount. As
 used herein, the term "therapeutically effective amount" means the total
 amount of the hydroxylated stilbene that is sufficient to show a
 meaningful benefit, i.e., treatment, healing, prevention, amelioration, or
 reduction in the symptoms of a herpesvirus infection, such as an HSV
 infection, or an increase in rate of healing, amelioration or reduction in
 the symptoms of such infection.
 By "treating" is meant curing or ameliorating a herpesvirus infection or
 tempering the severity of the infection. By preventing is meant blocking
 the formation of a primary lesion or recurrence of a lesion at the
 infected site. The dosages of the hydroxylated stilbene, particularly
 resveratrol, which can treat or prevent an HSV, VZV, or CMV infection,
 particularly an HSV infection, can be determined in view of this
 disclosure by one of ordinary skill in the art by rumiing routine trials
 with appropriate controls. Comparison of the appropriate treatment groups
 to the controls will indicate whether a particular dosage is effective in
 preventing or treating a virus infection at the levels used in a
 controlled challenge.
 It is contemplated that the various pharmaceutical compositions used to
 practice the method of the present invention should contain about 0.01
 .mu.g to about 10 mg, more preferably about 0.1 .mu. to about 1 mg, of the
 hydroxylated stilbene, most preferably from about 10 .mu.g to about 100
 .mu.g of resveratrol per/ml of the composition. Although a single
 application of the topical composition may be sufficient to ameliorate the
 pathological effects of the virus, it is expected that multiple doses will
 be preferred.
 DELIVERY
 Administration of the pharmaceutical composition is via local
 administration to the infected site. In those individuals who have
 experienced a primary lesion, it is preferred that the topical composition
 be applied at the prodromal stage of infection, i.e., during early
 symptoms of pain, tingling, parasthesia. Preferably, the composition is
 applied to the site of infection periodically, more preferably every three
 hours. The duration of therapy using the pharmaceutical composition of the
 present invention will vary, depending on the severity of the disease
 being treated and the condition and response of each individual patient.
 Ultimately the attending physician will decide on the appropriate duration
 of using the pharmaceutical composition of the present invention.
 The following examples of methods of using resveretrol to block formation
 of infectious HSV particles in vitro treating HSV infections are for
 purposes of illustration only and are not intended to limit the scope of
 the claims which are appended hereto.
 Example 1
 Inhibiting Formation of Infectious HSV-1 Particles by Treatment with
 Resveratrol
 A. Cultures of African green monkey kidney cells (Vero) cells, obtained
 from the American Type Culture Collection, Rockville, Md., were grown to
 confluence in Medium 199 supplemented with 5% fetal bovine serum, 0.075%
 NaHCO.sub.3, and 50 .mu.g/ml gentamycin sulfate in 25 cm.sup.2 tissue
 culture flasks. Cells were infected with HSV-1 at a multiplicity of
 infection (moi) of one and incubated at room temperature for one hour to
 allow for virus attachment to and penetration of the cell. Under these
 conditions, approximately half of the cells are infected with virus.
 Thereafter, the cultures were rinsed three time with media and incubated
 in medium containing resveratrol at a final concentration of 10 .mu.g/ml,
 25 .mu.g/ml, or 50 .mu.g/ml. Stock solutions of the resveratrol, obtained
 from Sigma Chemical Co, St. Louis, Mo. were prepared in 100% ethanol and
 diluted to the final concentration in tissue culture media. The
 concentration of alcohol in the medium ranged from 0.1 to 0.5%. Controls
 were treated identically, but were incubated without resveratrol.
 Upon addition of the medium to the cultures and at 24 hours time periods
 thereafter, i.e., 0 hours, 24 hours, 48 hours, and 72 hours after addition
 of the drug, cells and medium were frozen at -70.degree. C. Samples were
 then thawed, sonicated and titrated on Vero cells to determine the number
 of plaque forming units (pfu's) of virus produced by each culture.
 As shown in FIG. 1, the number of pfu's produced in the control cultures
 infected with an moi of 1 reaches peak production at approximately 48
 hours after infection. At this time, the system is exhausted, i.e., active
 virus has infected and destroyed not only those cells infected during the
 initial one hour of incubation but also those cells which became infected
 with virus released by the initially-infected cells. The lack of increase
 observed in the control cultures at 72 hours treatment indicates that the
 virus production has peaked, due to the lack of viable cells in which to
 reproduce.
 As shown in FIG. 1, treatment of cells with 50 .mu.g/ml of rveratrol
 inhibited formation of infectious virus particles in HSV-1 infected cells
 by more than 99% at 24 hours. By 72 hours, infectious HSV particles were
 virtually undetectable in cultures continuously incubated in the presence
 of 50 .mu.g/ml of resveratrol.
 These results also demonstrate that inhibition of virus replication by
 resveratrol is dose dependent. Treatment with 25 .mu.g/ml resveratrol
 reduced new virus production by only 95%, while treatment with 10 .mu.g/ml
 of resveratrol had little to no effect on formation of infectious virus
 particles.
 B. Cultures of human fibroblasts (MRC-5 cells) grown to confluence in
 Eagle's basal medium supplemented with 10% fetal bovine serum. 0.075%
 NaHCO.sub.3, and 50 .mu.g/ml gentamycin sulfate were infected with HSV-1
 at an moi of 1 as described above in part A. The infected cultures were
 incubated at 37.degree. C. for 72 hours in medium lacking resveratrol
 (control cultures) or containing 50 .mu.g/ml of resveratrol. The number of
 plaque-forming units produced in control cultures and cultures treated
 with resveratrol were determined as described above. The results indicated
 that treatment with 50 .mu.g/ml ml resveratrol inhibits formation of
 infectious virus particles by greater than 99% in the virus-infected human
 fibroblasts.
 Example 2
 Inhibiting Formation of Infectious HSV-2 Particles by Treatment with
 Resveratrol
 Cultures of Vero cells were infected with HSV-2 at an moi of 1 as described
 above in Example 1A and incubated for 72 hours in medium lacking
 resveratrol or containing 50 .mu.g/ml of resveratrol. The number of
 plaque-forming units produced control cultures and cultures treated with
 resveratrol were determined as described above. The results indicated that
 such treatment inhibits replication of HSV-2 in virally-infected cells by
 more than 99%.
 Example 3
 Inhibitinoz Formation of Infectious HSV-1 Particles by Contacting Cells
 with Resveratrol Prior to or Durino an Earlv Stage in Replication.
 Vero cell cultures were infected with HSV-1 as described above in Example
 1A except that the cells were infected with virus at an moi of 10. Under
 these conditions nearly all of the cells are infected with virus during
 the initial one hour incubation period. Following removal of unattached
 virus, the virally-infected cultures were incubated in control medium
 lacking resveratrol or medium to which resveratrol at a final
 concentration of 50 .mu.g/ml had been added at 1, 3, 6, or 9 hours after
 removal of the unattached virus. At 24 hours after infection, the number
 of pfu's present in the cells and medium of untreated and
 resveratrol-treated cultures was determined.
 The results presented in FIG. 2 demonstrate that resveratrol is most
 effective when administered to virally-infected cells during the early
 stages of viral replication. In cultures treated with resveratrol at one
 hour after infection, production of virus was reduced by more than 99%. In
 cultures treated with resveratrol at 3 or 6 hours after infection, the
 production of virus was inhibited by approximately 90%. When the
 hydroxylated stilbene was added 9 hours after infection, formation of
 infectious virus particles was not inhibited.
 To determine whether resveratrol blocks formation of infectious herpes
 virus particles by directly inactivating the virus, a standard inoculum of
 HSV-1 was mixed with 10 or 50 .mu.g/ml of resveratrol in medium, with 0.5%
 ethanol in medium, or with media alone and placed at room temperature. The
 number of residual pfti's present at 1, 10, 30, and 60 minutes after
 addition of each respective solution to the virus was determined by plaque
 assay. The results demonstrated that resveratrol did not directly
 inactivate HSV.
 Example 4
 Inhibiting Formation of Infectious HSV-2 Particles by Contacting Cells with
 Resveratrol Prior to or During an Early Stage in Replication.
 Vero cells were infected with HSV-2 at an moi of 10 and the effect of
 treatment with resveratrol at 1, 3, 6, or 9 hours after removal of the
 unattached virus was determined as described in Example 3. The results
 were the same. Administration of resveratrol to the cells within the first
 6 hours after infection blocked replication of HSV-2, while treatment with
 the hydroxylated stilbene at 9 hours after infection had little to no
 effect on formation of infectious HSV-2 particles.
 Example 5.
 Inhibiting HSV Replication
 Vero cells were grown to confluence and infected with HSV-1 at an moi of 1
 and then incubated in media lacking resveratrol (control cultures) or in
 media containing resveratrol at a concentration of 50 .mu.g/ml. One set of
 infected cells was maintained in resveratrol for a period of 72 hours. In
 another set of infected cells the resveratrol-containing media was
 replaced with media lacking resveratrol at 24 hours. In another set of
 cells the resveratrol-containing media was replaced with media lacking
 resveratrol at 48 hours after infection. The number of infectious HSV
 particles produced by each set of infected cells was determined by plaque
 assay.
 The results shown in FIG. 3 demonstrate that the inhibitory effect of
 resveratrol on HSV replication in virus infected cells is reversible.
 Accordingly, continuous treatment of HSV-infected cells with resveratrol
 maintains the virus in a non-infectious state. Discontinuing the
 resveratrol treatment allows replication of the virus to proceed in what
 appears to be a normal fashion. The same results were obtained with HSV-2
 (data not shown). The results presented in FIG. 3 also suggest that HSV
 replication in the resveratrol treated cells was blocked at an early
 phase, i.e., replication of HSV had not progressed past the stage where
 cells are so damaged that they are unable to support replication of this
 herpes virus.
 The results presented in FIG. 3 also indicate that exposure of mammalian
 cells to 50 .mu.g/ml resveratrol for a prolonged period of time does not
 kill the cells. Cell viability studies confirmed that treatment of
 uninfected Vero cells with 50.mu.g/ml resveratrol for 24 hours was not
 toxic. When uninfected Vero cells were incubated in medium containing 100
 .mu.g/ml resveratrol for 24 hours, an 18% reduction in Vero cell viability
 was observed.
 Example 6
 Characterizing Viral Proteins Produced in the Presence of Resveratrol
 ICP-4 is an immediate-early regulatory protein of HSV-1 that is required
 for efficient replication of this virus. To determine whether ICP-4
 production is altered by treatment with resveratrol, Vero cells were
 infected with HSV-1 at an moi of 1 and incubated in control medium or
 medium containing 50 .mu.g/ml resveratrol for 24 hours. Infected cells
 were scraped from the flask, collected by centrifugation, and resuspended
 in cold tris-buffered saline, pelleted by centrifugation, and the cell
 pellet frozen at -70.degree. C. Proteins were extracted from the thawed
 pellets, separated by 6-15% SDS-PAGE, and assayed on a Western blot by
 reacting with mouse monoclonal antibody to ICP-4 from Goodwin Institute
 for Cancer Research Inc., Fl.
 As shown in FIG. 4, treatment of HSV-infected cells with resveratrol
 significantly reduced synthesis of ICP-4, a major regulatory protein.
 These results confirm that treatment with resveratrol inhibits synthesis
 herpes viruses at an early phase in the replicative scheme. These results
 also indicate that cultured cells treated with reseveratrol are a useful
 model system for characterizing the herpes virus gene products that are
 made during the immediate early phase and early phase of HSV replication.
 Example 7
 Maintaining HSV in a Latent Stage in Organ Culture.
 SJH1 (Charles Rivers Laboratory) mice were anesthetized with metofane and
 both corneas lightly scratched with a 30 gauge needle. A 50 .mu.g inoculum
 of 10.sup.4 pfu of HSV-1 was placed on the eye surface which was then
 closed and gently massaged. The animals were rested for not less than 30
 days. The animals were euthanized with CO.sub.2, and the left and right
 trigeminal ganglia harvested. The ganglia were aseptically removed and
 placed in separate wells of a 12 well tissue culture plate containing
 tissue culture media with 50 .mu.g/ml of resveratrol or without
 resveratrol. On each day of the following 10 days, the fluid from each
 well was collected, frozen, and replaced with fresh media. At the end of
 ten days, each sample of collected medium was tested for the presence or
 absence of infectious virus usinig a plaque assay.
 The results are shown in Table 1 below.
 TABLE 1
 The Effect of Resveratrol on Reactivation of HSV from Latently-infected
 Trigeminal Ganglia
 Number Reactivated.sup.1
 Left Ganglia - No Resveratrol Right Ganglia - With Resveratrol
 8 0
 .sup.1 Left and right trigeminal ganglia were removed from each mouse and
 incubated for ten days in media with or without resveratrol. Samples were
 taken and tested daily for infectious virus. A positive result is the
 appearance of infectious virus in any of the ten day test samples.
 As shown in Table 1, ganglia from 8 of the animals released infectious
 virus when incubated in medium lacking resveratrol, while no virus was
 released from the corresponding ganglia that had been incubated in medium
 containing resveratrol. These results indicate that organ cultures
 incubated in medium containing resveratrol are good model systems for
 studying latency of herpesvirus.
 Example 8
 Treating HSV Infections with a Topical Composition Comprising Resveratrol.
 Female SKHI hairless mice were obtained from Charles River Laboratory. The
 animals were anesthetized with metofane and lightly scratched once on
 their back with a 30 gauge needle. A cotton swab saturated with HSV-1, at
 a concentration of 10.sup.7 pfu/ml in media, was applied to the scratch
 for a few seconds. The animals were returned to their cage and, after one
 hour, were treated with resveratrol by soaking a cotton swab in
 resveratrol at a concentration of 50 .mu.g/ml in Media 199 and applying it
 to the scratch site for several seconds. The virus-infected site was
 treated with the resveratrol containing solution as described above three
 times a day. The animals were monitored daily for lesion formation
 according to the following schedule: 0 =no lesion; 1+=erythema;
 2+=erythema, limited vesicle formation 3+=erythema, moderate vesicle
 formation, few ulcers; and 4+=erythema, extensive vesicle formation,
 multiple ulcers, scab formation. The results are shown in Table 2 below.
 TABLE 2
 Effect of Resveratrol on HSV-1 Lesion Formation in the Skin of Hairless
 Mice
 Day 5 Day 8 Day 9 Day 10 Day 11 Day 12
 Day 15
 L.sup.3 R L R L R L R L R
 L R L R
 Resveratrol- .7+.sup.4 0 3+ 0 2.3+ .33+ 2.7+ 1.3+ 2.3+ 1+ 1.7+ 1+
 .33+ 0
 Treated.sup.1
 Control.sup.2 1+ 0 3+ 2+ 4+ 3+ 4+ 3+ 4+ 3+ 2+ 1+ 0 0
 .sup.1 Average of three animals.
 .sup.2 One animal.
 .sup.3 L = left side; R = right side.
 .sup.4 0 = no lesions; 1+ = erythema; 2+ = erythema, limited vesicle
 formation; 3+ = erythema, moderate vesicle formation, few ulcers; 4+ =
 erythema, extensive vesicle formation, multiple ulcers, scab formation.
 As shown in Table 2, HSV1-infected control animals and HSV-1 infected
 animals treated with resveratrol as described above began showing signs of
 infection five days after being exposed to the virus. Treatment with
 resveratrol was stopped at that time for two days to allow lesions to
 fully develop. Lesions first began to appear on the left side of the
 animal and then spread to the right side. In both resveratrol-treated and
 control animals, lesions developed on both sides, but lesion development
 in resveratrol-treated animals appeared to lag behind the control in both
 time of appearance and extent of tissue damage. Both groups of animals
 appeared to recover at about 15 days after exposure to the virus. These
 results demonstrate that treatment with resveratrol reduces the severity
 of HSV-1 infections.