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Latent Herpes Simplex Virus Infections in Sensory Ganglia of Hairless Mice Prevented by Acycloguanosine Richard J. Klein, Alvin E. Friedman-Kien and Eugene DeStefano Antimicrob. Agents Chemother. 1979, 15(5):723. DOI: 10.1128/AAC.15.5.723. Downloaded from http://aac.asm.org/ on November 12, 2011 by guest Updated information and services can be found at: http://aac.asm.org/content/15/5/723 These include: CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://aac.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1979, p. 723-729 Vol. 15, No. 5 0066-4804/79/05-0723/07$02.00/0 Latent Herpes Simplex Virus Infections in Sensory Ganglia of Hairless Mice Prevented by Acycloguanosinet RICHARD J. KLEIN,* ALVIN E. FRIEDMAN-KIEN, AND EUGENE DESTEFANO Department ofMicrobiology, New York University School of Medicine, New York, New York 10016 Received for publication 26 February 1979 Acycloguanosine (ACG) was able to prevent the fatal outcome of herpes Downloaded from http://aac.asm.org/ on November 12, 2011 by guest simplex virus-induced skin infections of the lumbosacral or orofacila area in hairless mice. Topical ACG treatment was more effective than systemic treatment in preventing the evolution of skin lesions. Acute ganglionic infections in the trigeminal ganglia were prevented by ACG, and latent ganglionic infections did not become established when the ACG treatment was initiated 3 h after infection. Serum antibody titers were, on the average, eight times higher in mice which developed latent ganglionic infections after ACG treatment than in mice without evidence of herpes simplex virus latency in ganglia. Reinoculation of ACG-treated mice at a site different from that of the primary inoculation did not lead to the establishment of a second latent infection with the homologous virus type when a latent infection was already present. In mice without evidence of latent infection after the primary inoculation, a latent infection at the site of reinoculation became established in 25% of the animals. It has been recently shown by Elion et al. (1) In the present study we have evaluated the that a guanine derivative with an acyclic side efficacy of ACG in HSV-induced skin infections chain, 9-(2-hydroxyethyoxymethyl) -guanine in hairless mice. The evaluation was based upon (acycloguanosine [ACG]), is a potent in vitro the effect on skin lesions, the mean survival inhibitor of herpes simplex virus type 1 (HSV- time, and the mortality rate. The pathogenesis 1). In HSV-infected cells ACG is converted to of the HSV-induced skin infection during the its triphosphate, which acts as a selective inhib- treatment and the specific antibody response itor of HSV deoxyribonucleic acid nucleotidyl- were likewise investigated. As we have pointed transferase. ACG serves as a substrate for HSV- out in a previous study (3), evaluation of the specific thymidine kinase; cellular thymidine ki- effectiveness of any chemotherapeutic agent ac- nase which occurs naturally in uninfected cells tive against HSV infection should include the does not effectively utilize ACG as a substrate. determination of its ability to prevent the estab- Subsequent studies (8) have shown that ACG, lishment of latent infections in the sensory gan- on a molar basis, is about 10 times more potent glia after the primary infection. It was shown than 5-iodo-2'-deoxyuridine, 160 times more ac- that PAA affords good protection against the tive than adenine arabinoside (Ara-A), and establishment of latent infections in sensory gan- about 600 times more active than phosphono- glia (5, 6, 12), whereas Ara-A and Ara-A-mono- acetic acid (PAA). ACG was tested for activity phosphate are much less effective (3, 6). Trifluo- in mice infected intracerebrally with HSV: given rothymidine and Ara-tri-O-acetate do not dis- by the oral route or subcutaneously in a dose of play any protection (A. E. Friedman-Kien and 100 mg/kg of body weight twice daily for 5 days, R. J. Klein, Abstr. Annu. Meet. Am. Soc. Micro- the compound increased the survival time of biol. 1978, A38, p. 7). We therefore investigated infected mice (8). ACG ointments were also eval- the ability of ACG to prevent the establishment uated in experimental herpetic keratitis in rab- of latent ganglionic infection in the experiments bits and in HSV-induced lesions in guinea pigs. described below. In both models, the substance conferred good protection against the infections (2, 8). No quan- MATERLALS AND METHODS titative data regarding the skin infections were Virus. The S strain of HSV-1, used throughout this given in the above-mentioned experiments. experiment, and the Gordon strain of HSV-2 have t Publication no. 40 from the Cooperative Antiviral Testing been described previously (4, 5). Group of the Antiviral Substances Program, Development Inoculation of mice. Two skin sites were used for and Applications Branch, National Institute of Allergy and virus inoculation: infection of the lumbosacral area, Infectious Diseases, National Institutes of Health. associated with a high mortality rate (60 to 100%) and 723 724 KLEIN, FRIEDMAN-KIEN, AND DESTEFANO ANTIMICROB. AGENTS CHEMOTHER. severe skin lesions, and infection of the orofacial area, from 0 to 4) (Table 1). The 50-mg/kg dose associated with low mortality (O to 40%) and less conferred better protection than did the 20-mg/ severe skin lesions. Each area was infected by rubbing kg dose. All but one of the ACG-treated mice the scarified skin site with a virus suspension contain- survived the infection, whereas the mortality ing 106 plaque-forming units per ml, as determined by a plaque assay on Vero cells. The frequency of detect- rates in placebo-treated mice were 50% in those able latent HSV infection after lumbosacral inocula- inoculated in the orofacial area and 62% in those tion is about 60 to 70% in the spinal ganglia of surviving inoculated in the lumbosacral region. Both the untreated or placebo-treated mice (3, 5), whereas the high and the low doses were equally effective in frequency of latent infections in the trigeminal ganglia preventing the fatal outcome of the infection. of orofacially inoculated mice approaches 100% (4, 6). However, the systemic treatment did not sig- Therefore, the effect of antiviral treatments on the nificantly prevent the eventual establishment of Downloaded from http://aac.asm.org/ on November 12, 2011 by guest mortality rate and severity of skin lesions was esti- latent HSV infection in the spinal ganglia of the mated from lumbosacrally inoculated mice, and the surviving mice. frequency of latent infections after treatment of the primary infection was estimated from mice inoculated Evolution of skin lesions, mortality rate, in the orofacial area. and latent ganglionic infections after topi- Procedures for cocultivation of mouse ganglia, se- cal treatment with ACG. Hairless mice were rum HSV-specific antibody titration, and monitoring inoculated percutaneously in the lumbosacral or the establishment of acute and chronic virus infections orofacial skin area with HSV-1. ACG gels or in the ganglia have been described in previous publi- ointments at a concentration of 1 or 5% were cations (3, 5, 6). applied four times daily over a period of 5 days Antiviral compounds. ACG was used as 1 and 5% or less. Treatments were initiated at various gels and creams (Weilcome Research Laboratories, intervals after virus inoculation. Research Triangle Park, N.C.). The composition of In mice inoculated in the lumbosacral area the cream base was polyethylene glycol 300 (liquid) and polyethylene glycol 1500 (solid). The composition the 5% gel and ointment almost completely pre- of the gel, which has a tendency to separate upon vented the development of skin lesions when the prolonged storage, was sodium alginate, glycerol and treatment was initiated 3 h after virus inocula- methylparaben. tion. Only mild lesions, which healed rapidly, were observed when mice were treated with the RESULTS 1% gel or ointment (Fig.1). The 5% gel applied Evolution of skin lesions, mortality rate, 24 and 48 h after virus inoculation curtailed the and latent ganglionic infections after sys- development of skin lesions, and the lesions al- temic treatment with ACG. Hairless mice ready present healed rapidly (Fig. 2). Similar were inoculated percutaneously in the lumbo- results were observed when topical treatment sacral or orofacial area with HSV-1. Three hours with the 5% ointment was initiated 24 h after after virus inoculation, treatment with ACG was virus inoculation. All mice treated with the 1% initiated. Two different dosages, (20 and 50 mg/ or 5% ACG gel and ointment according to var- kg) were given intraperitoneally in two daily ious schedules survived. The observed mortality injections over a period of 4 days (total dosages, rate in parallel placebo-treated mice was 100% 80 and 200 mg/kg, respectively). (Table 2). ACG reduced significantly the average inten- Latent infections in the spinal ganglia were sity of the skin lesions and the frequency of not detected in mice treated 3 h after infection severe skin lesions (lesion score of >2 on a scale with either the 1% or the 5% ointment. When TABLE 1. Systemic treatment with ACG of HSV skin infection in hairless mice Route of inoculation Treatment' Avg intensity of skin Frequency of severe lesions' lesions (P') Mority rate (P") Latent ganglionic in- fections (P') Orofacial Placebo 3.50 8/8 4/8 4/4 20 mg/kg 1.81 5/8 (NS") 1/8 (NS) 6/7 (NS) 50 mg/kg 0.12 0/6 (<0.001) 0/6 (NS) NTe Lumbosacral Placebo 3.75 8/8 5/8 3/3 20 mg/kg 1.20 3/8 (<0.02) 0/8 (<0.02) 3/7 (NS) 50 mg/kg 0.44 0/7 (<0.001) 0/7 (<0.02) 3/7 (NS) Daily dose of ACG given in two intraperitoneal injections over a period of 4 days; placebo treatment consisted of two daily intraperitoneal inoculations of 0.5 ml of phosphate-buffered saline. 'Graded on a scale from 0 to 4. c Fisher exact test. d NS, Statistically not significant. 'NT, Not tested. VOL. 15, 1979 HSV LATENCY PREVENTED BY ACYCLOGUANOSINE 725 4 from the establishment of latent infections, 0 whereas the same preparation given for only 3 t) 3 flofl SUrViVg days or initiated after a delay of 24 h protected C 0 St \ t only 30% of the mice (Table 3). The difference .0 was statistically significant (P < 0.04). Latent HSV infections in the trigeminal ganglia were 2 observed in 70% of the mice treated with 1% 1._- 4 o _* / o all surviving ACG ointment but only in 20% of the mice *.XtX V treated with 5% ACG ointment (P < 0.04). With ,% IZ ,,, comparable drug concentrations, gels appeared 1 I 3 5 7 9 11 13 to be somewhat more effective than ointments: Days after infection although the differences were not statistically Downloaded from http://aac.asm.org/ on November 12, 2011 by guest FII.G. 1. Evolution of HSV-induced skin lesions in significant the frequency of latent infections in . the 1!umbosacral area of hairless mice treated with the trigeminal ganglia in comparable treatment ACG,. The compound was applied topically four times groups was almost always higher in ointment- dail v over a period of 5 days. Symbols: 0, untreated; treated than in gel-treated mice. 0, tri*eated with 1% gel; O, treated with 1% ointment Relationship between presence of latent (lOimiice per group). HSV infections in the trigeminal ganglia and HSV-specific serum antibody titers. Mice inoculated in the orofacial area and treated r_ * 0 *with ACG or placebo gels were killed 3 to 4 0 ^ A weeks after inoculation, by heart puncture. Se- 0 .2 c0 none surviving rum antibody titers were determined by quantal neutralization in microplates. Latent infections - were detected by cocultivation of trigeminal gan- glion fragments in the presence of virus-sensitive 0o all surviving human foreskin cells as previously described (6). o - V Despite a relatively wide range of antibody 4 jy ,0_& _ Vtiters in individual mice from the different treat- ment groups, the highest antibody titers were 3 5 7 9 11 13 associated with the presence of latent infections in the trigeminal ganglia (Fig. 3). In treated mice without evidence of latent infections, antibody FI4G. 2. Evolution of HSV-induced skin lesions in titers were low or below our detection levels, and the 1 'umbosacral area of hairless mice treated topi- titers higher than 102 but less than 1025 were cally with a 5% ACG gel. The gel was applied four seen only in 25% of the mice. On the other hand, timess daily over a period of 5 days. Symbols: 0, titers of 1025 or higher were observed in 58% of plac ebo-treated mice; 0, treatment initiated24 h after the mice with latent infection in the trigeminal virus inoculation; @. treatment initiated 48 h after ganglia. The mean antibody titer in 29 mice with virus latent ganglionic infections was 2.58 ± 0.52 logio units, whereas in those without evidence of la- the ttreatment was delayed for 24 h after virus tent infections the mean titer was 1.65 ± 0.45 inoci ulation, the frequency of latent infections in log10 units, i.e., an 8.5-fold difference. the spinal ganglia of mice was reduced, but Pathogenesis of orofacial HSV infection latenit infections could not be completely pre- during topical ACG treatment. Mice inocu- vent4 ed. lated in the orofacial area with HSV-1 were Si,milar results were observed after the treat- treated with placebo and 1 and 5% ACG oint- mentt of the orofacial infection with 1 and 5% ments, starting the applications 3 h after infec- ACG: gels and ointments. The development of tion. The topical treatment was given four times skin lesions and the mortality among mice daily for up to 5 days. Groups of four mice were treatWed according to the various schedules de- killed 2, 4, 7, 10, and 21 days after infection. The scribted were completely prevented (Table 3). trigeminal ganglia were dissected, minced, and The drug concentration in the preparation and homogenized. After three cycles of freeze-thaw- the dlelay in initiation and duration of the treat- ing, the suspension was centrifuged and tested mentt had, however, a definite effect on the fre- for the presence of virus by a plaque assay on quency of latent HSV infections established in Vero cells. Ganglion fragments obtained 21 days the tirigeminal ganglia (Table 3). Treatment with after infection were cocultivated in the presence 1% ALCG gel initiated 3 h after virus inoculation of virus-sensitive human foreskin cells. and jgiven for 5 days protected 80% of the mice In placebo-treated mice, free virus was de- 726 KLEIN, FRIEDMAN-KIEN, AND DESTEFANO ANTIMICROB. AGENTS CHEMOTHER. TABLE 2. Effectiveness ofACG in topical treatment of HSV-induced skin infections in the lumbosacral area of hairless mice Initiation of treatment A h Frequency of latent infec- T1'reatment (in h postinfection)a AvglesionscoreMortality rate tions Gel Placebo 3 3.60 10/10 ACG 1% 3 1.00 0/10 4/10 24 1.05 0/10 NT' ACG 5% 3 0.15 0/10 0/10 Downloaded from http://aac.asm.org/ on November 12, 2011 by guest 24 0.65 0/10 2/10 48 1.45 0/10 5/10 Ointment Placebo 3 4.00 10/10 ACG 1% 3 0.65 0/10 0/10 24 0.85 0/10 4/9 ACG 5% 3 0.05 0/10 0/10 24 0.10 0/10 4/10 " Treatments were applied four times daily for 5 days. b Graded on a scale from 0 to 4. 'NT, Not tested. TABLE 3. Effectiveness of ACG in topical treatment of HSV-induced skin infections in the orofacial area of hairless mice ti Treatment Treatment schedule Avg lesion scorea Mortality rate fection Gel Placebo 3 h p.i.,c 5 days 3.65 2/7 5/5 ACG 1% 3 h p.i., 5 days 0.20 0/10 2/10" 3 h p.i., 3 days 0.25 0/10 7/10 24 h p.i., 5 days 0.20 0/10 7/10 ACG 5% 3 h p.i., 5 days 0.00 0/10 0/10" 3 h p.i., 3 days 0.00 1/10w 1/9" 24 h p.i., 5 days 0.20 0/10 2/10 48 h p.i., 5 days 0.25 1/10t 5/9 Ointment Placebo 3 h p.i., 5 days 3.90 4/10 6/6 ACG 1% 3 h p.i., 5 days 0.25 0/10 7/10 ACG 5% 3 h p.i., 5 days 0.10 0/10 2/10" 24 h p.i., 5 days 0.10 0/10 6/10 a Graded on a scale from 0 to 4. b Detected by cocultivation of trigeminal ganglion fragments in the presence of HSV-sensitive human foreskin cells. c p.i., Postinfection. d The probability that the reduced number of latent HSV infections in the trigeminal is due to chance is less than 0.01. e Death not a result of the HSV infection. tected in ganglionic homogenates 2, 4, and 7 free virus was not detected in ganglionic homog- days after infection, and latent infections were enates, with the exception of one mouse treated observed in all ganglion fragments cocultivated with 5% ACG ointment in which 2 plaque-form- 21 days after infection. In ACG-treated mice, ing units were detected in the homogenate pre- VOL. 15, 1979 HSV LATENCY PREVENTED BY ACYCLOGUANOSINE 727 @ 4.0 Latent infections present glia. Only 1 mouse was found to have latent 37 3 O obsent 0 0 HSV infections in the spinal ganglia (established 3 4 after the primary infection in the lumbosacral c 3 area) and in the trigeminal ganglia (established E, c; S8 2 after reinoculation in the orofacial region). Neu- * 0 00 C tralization with HSV type-specific rabbit anti- i, 022 sera showed that the virus reactivated from the : , 2.2 spinal ganglia was HSV-1, whereas the virus isolated from the trigeminal ganglia was HSV-2. 3 3hpi 3h pi 24hpi 48hpi 3hpi 3hpi 24hpi S days 3days 5doys Sdays 15doys 3days Sdoys 3 h pti Sdays DISCUSSION Our results indicate that HSV-induced skin Downloaded from http://aac.asm.org/ on November 12, 2011 by guest Acycloguanosine Acycloguanosine pIGzcebo 5% gel 1% gel gel infections in hairless mice can be successfully FIG. 3. Relationship between thepresence of latent treated topically with ACG gels and ointments. HSV infection in the trigeminal ganglia after ACG Similar results have been presented by Schaeffer treatment and HSV-specific serum antibody titers. et al. (8) in skin infections of guinea pigs. When Bars represent the mean antibody titer of indicated a 1% ACG ointment was applied twice daily for treatment group. Treatment was applied four times 3 days on the percutaneously infected guinea daily for indicated number of days. p.i., Postinfection. pigs, the treated sites healed in about 3 days, whereas on the untreated sites the lesions were pared 7 days after virus inoculation. One of the still in progress (8). However, HSV infections in four mice from each group treated with 1 or 5% guinea pigs are mild, and central nervous system ACG ointment showed evidence of latent infec- involvement with a fatal outcome is a rare event tions in ganglion fragments obtained 21 days (10). E. R. Kern, J. T. Richards, J. C. Overall, after infection. The results are summarized in Jr., and L. A. Glasgow (Program Abstr. Intersci. Table 4. Conf. Antimicrob. Agents Chemother., 18th, At- We have noted that the virus reactivation lanta, Ga., Abstr. no. 264, 1978) have shown that time, i.e., the time elapsed from the initiation of mice inoculated intravaginally with HSV-2 and ganglion cultures until the appearance of the treated with ACG showed a statistically signifi- virus-induced cytopathic effect, was 10 days for cant reduction in the intensity of vaginal lesions, ganglia from the two ACG-treated mice, whereas a decrease of the mortality rate, and a reduction it required only 5 days for the ganglia of the four of virus shedding from the vaginal lesions. No placebo-treated mice. This was not an isolated data were provided regarding the ability of the observation, since the virus reactivation time ACG to prevent the establishment of latent HSV from latently infected ganglia of ACG-treated infections in the sensory ganglia of the guinea mice was consistently longer than the virus reac- pigs or mice. tivation time from ganglia of placebo-treated TABLE 4. Acute and latent HSV infections in mice. In ganglia of placebo-treated mice the trigeminal ganglia ofplacebo- and ACG-treated mean virus reactivation time was 4 days (range, mice 3 to 5 days), whereas in ACG-treated mice this No. of mice with virus in trigeminal interval increased to almost 8 days (range, 4 to ganglia' at: 10 days) (Table 5). Treatment Resistance to reinfection of ACG-treated 10 21 2 days 4 days 7 days days days mice. Groups of mice inoculated in the lumbo- p.i. p.i. sacral area with HSV-1 and treated with ACG were reinoculated 3 to 4 weeks later in the Placebo 1/4 4/4 3/4 0/4 4/4 orofacial region with HSV-1 or HSV-2 (Table 6). ACG 1% oint- 0/4 0/4 0/4 0/4 1/4 No lesions were observed after reinoculation in ment mice treated systemically, but 8 out of the 19 ACG 5% oint- 0/4 0/4 1/4 0/4 1/4 ment mice treated topically developed small lesions. In 7 out of the 34 reinoculated mice a latent a Numerator = number of tested mice; denominator infection became established in the trigeminal = number of mice with evidence of acute (2, 4, 7, and ganglia (Table 6). The 5% ACG gel-treated mice 10 days) or latent (21 days) HSV infections. Free virus with latent infections in the trigeminal ganglia detected in ganglion homogenates prepared promptly after the killing of mice was indicative of an acute were among those which developed lesions after infection. Appearance of cytopathic effect in coculti- reinoculation. Six latent infections in the trigem- vated ganglion fragments was a manifestation of reac- inal ganglia became established in mice without tivated latent virus. b evidence of latent infections in the spinal gan- p.i., Postinfection. 728 KLEIN, FRIEDMAN-KIEN, AND DESTEFANO ANTIMICROB. AGENTS CHEMOTHER. TABLE 5. Virus reactivation time in cocultivated trigeminal ganglia latently infected with HSV after treatment with ACG gelsa Reactivation time (days) Gel Treatment schedule Observed latent infec- tions Mean ± standard devia- tion Range Placebo 3 h p.i.,b 5 days 5 4.00 ± 0.71 3-5 ACG 1% 3 h p.i., 3 days 7 6.00 ± 1.41 4-8 ACG 1% 24 h p.i., 5 days 7 7.14 ± 2.12 4-10 ACG 5% 48 h p.i., 5 days 5 7.60 ± 2.30 4-10 a From groups of mice in Table 3. Downloaded from http://aac.asm.org/ on November 12, 2011 by guest b p.i., Postinfection. TABLE G. Latent ganglionic infections in ACG-treated hairless mice after primary infection in the lumbosacral area with HSV-I and reinoculation at a distant site (orofacial area) Latent infection Treatment' Reinoculationh No. of mice Spinal Trigeminal Spinal + trigeminal None HSV-2 3 2 0 1' 20mgi.p. HSV-2 4 1 0 0 50mgi.p. HSV-2 8 3 1 0 1% gel HSV-1 9 4 2 0 5% gel HSV-1 10 0 3 0 a The intraperitoneal (i.p.) treatment was given twice daily over a period of 5 days (20 or 50 mg/kg per day); the topical treatment was applied four times daily over a period of 5 days. All treatments were initiated 3 h after the primary infection. b Reinoculation was performed 3 to 4 weeks after the primary infection. HSV from the spinal ganglia was of type 1, and that from the trigeminal ganglia was of type 2, as determined by neutralization with type-specific rabbit sera. Our results show that topically applied ACG prevent both skin lesions and the eventual de- preparations given as late as 48 h after virus velopment of latent infections in sensory ganglia inoculation in the lumbosacral or orofacial area, (5, 6, 12). However, the effectiveness of PAA when lesions are already present, can prevent decreased sharply when the treatment was de- their further evolution. Although the mortality layed, and the compound also had an irritating rate among placebo-treated mice was 100%, none effect on the skin. No such irritations were ob- of the ACG-treated mice died, even when the served in ACG-treated mice. treatment was delayed for 48 h after virus inoc- The results obtained with ACG treatment ulation in the lumbosacral area. It appears also substantiated our observations made with Ara- that when ACG treatment is applied 3 h after A, Ara-A-monophosphate, and PAA that the infection, no latent HSV infections are estab- orofacial route of inoculation provides a reliable lished in the sensory ganglia. However, when estimate of the ability of an antiviral compound the treatment was delayed or when the concen- to prevent the establishment of latent HSV in- tration of the drug in the ointments was de- fections in the trigeminal ganglia (6). As opposed creased, an increase in the frequency of latent to the orofacial route of inoculation, the lumbo- ganglionic infections was observed. This shows sacral route yields somewhat irregular responses that ACG has little effect on the virus which has due to the fact that latent infections can be already invaded the ganglion. detected only in 60 to 70% of the untreated mice Compared to other antiviral agents, ACG surviving the viral infection. However, the latter seems to be the most effective and least toxic route of inoculation is a critical test in estimating compound in the treatment of HSV skin infec- the ability of an antiviral agent active against tions in mice which we have evaluated to date. HSV to prevent the fatal outcome of the skin We have shown that Ara-A and Ara-A-mono- infection. phosphate, applied topically, can reduce the in- The experiments described showed that topi- tensity of skin lesions and reduce significantly cal treatment with ACG prevented virus pene- the mortality rate, but cannot prevent the estab- tration of cutaneous nerve endings, since no free lishment of latent HSV infections in the spinal virus was generally detected in trigeminal gan- or trigeminal ganglia (3, 6). Among the tested glion homogenates. We have observed a rela- antiviral agents, only PAA had the ability to tionship between the amount of free virus in the VOL. 15, 1979 HSV LATENCY PREVENTED BY ACYCLOGUANOSINE 729 ganglia during the acute phase of the infection latent infections in the spinal ganglia, 12 dis- and the virus reactivation time in cocultivated played latent infections in trigeminal ganglia latently infected ganglia (6). When ACG-treated (26%), whereas in 19 mice with latent HSV in mice develop latent ganglionic infection, the vi- the spinal ganglia, only 1 was detected with a rus reactivation time is about twice as long as latent infection in the trigeminal ganglion (5%). that found in the ganglia of placebo-treated The difference is statistically significant (P < mice. Our data do not provide information as to 0.05) and indicates that double latent ganglionic the actual number of latently infected cells in HSV infections in mice are a rare event. the ganglia, but Walz et al. (11) have shown that about 0.1% of the ganglionic cells are infected ACKNOWLEDGMENTS and that if the immune response of the mouse inhibits viral replication in the skin, the number We acknowledge the excellent technical assistance of Eileen Downloaded from http://aac.asm.org/ on November 12, 2011 by guest of cells becoming infected would be reduced Brady and the voluntary help of Mark Gratewohl. This work was supported by Public Health Service (PHS) grant DE- correspondingly. This conclusion may be ex- 04958 from the National Institute of Dental Research and by tended to include drug treatments which inhibit PHS contract NO1-02131 from the National Institute of Al- virus replication in the skin, thus reducing the lergy and Infectious Diseases. number of ganglionic cells which become la- tently infected. Since ganglia from treated mice LITERATURE CITED are cocultivated in fragments, it is easily con- ceivable that virus reactivated in a few cells, 1. Elion, G. B., P. A. Furman, J. A. Fyfe, P. DeMiranda, L. Beauchamp, and H. J. Schaeffer. 1977. Selectivity some of them located deep inside the fragment, of action of an antiherpetic agent, 9-(2-hydroxyethoxy- may require a longer time to be released and methyl) guanine. Proc. Natl. Acad. Sci. U.S.A. 74:5716- reach the indicator cells where the cytopathic 5720. effect is observed than would virus reactivated 2. Kaufman, H. E. 1978. Herpetic keratitis. Invest. Oph- thalmol. Visual Sci. 17:941-957. from ganglia of untreated mice, containing 10 to 3. Klein, R. J., and A. E. Friedman-Kien. 1977. Latent 100 times more latently infected cells. herpes simplex virus infections in sensory ganglia of Mice in which treatment with ACG was initi- mice after topical treatment with adenine arabinoside ated 3 h after infection had low or undetectable and adenine arabinoside monophosphate. Antimicrob. levels of serum antibodies. It appears from our Agents Chemother. 12:577-581. 4. Klein, R. J., A. E. Friedman-Kien, and E. Brady. 1978. results that some of these mice can be reinfected Latent herpes simplex virus in ganglia of mice after with subsequent establishment of a latent gan- primary infection and reinoculation at a distant site. glionic infection if the reinoculation is performed Arch. Virol. 57:161-166. 5. Klein, R. J., A. E. Friedman-Kien, A. A. Fondak, and at a site distant from that of the primary infec- E. Buimovici-Klein. 1977. Immune response and la- tion. Similar results were obtained previously in tent infection after topical treatment of herpes simplex mice reinoculated at a different site after PAA virus infection in hairless mice. Infect. Immun. 16:842- treatment of the primary infection (4). In mice 848. with latent infections in the ganglia innervating 6. Klein, R. J., A. E. Friedman-Kien, and P. B. Yellin. 1978. Orofacial herpes simplex virus infection in hairless the site of primary infection, the establishment mice: latent virus in trigeminal ganglia after topical of latent infections in the ganglia innervating the antiviral treatment. Infect. Immun. 20:130-135. area of reinoculation was prevented. In the pres- 7. McKendall, R. R. 1977. Efficacy of herpes simplex virus ent experiments, only 1 out of the 10 mice with type 1 immunization in protecting against acute and latent infection by herpes simplex virus type 2 in mice. latent infections in the spinal ganglia developed Infect. Immun. 16:717-719. a latent infection in the trigeminal ganglia after 8. Schaeffer, H. J., L. Beauchamp, P. deMiranda, G. B. reinoculation with the heterologous virus type. Elion, D. J. Bauer, and P. Collins. 1978. 9-(2-hydrox- ethoxymethyl) guanine activity against viruses of the Although McKendall (7) has shown that im- herpes group. Nature (London) 272:583-585. munity to HSV-1 in mice protects against acute 9. Shavrina Asher, L. V., M. A. Walz, and A. L. Notkins. and latent infections with HSV-2, it appears that 1978. Effect of immunization on the development of protection is not absolute. Recently, it was latent ganglionic infection in mice challenged intrava- shown (9) that in mice immunized with HSV-1, ginally with herpes simplex virus type 1 and 2. Am. J. Obstet. Gynecol. 131:788-791. 40% of the mice surviving the vaginal challenge 10. Tenser, R. B., and G. D. Hsiung. 1977. Pathogenesis of with HSV-2 developed latent ganglionic infec- latent herpes simplex virus infection in the trigeminal tions. This relatively low protection rate can be ganglion in guinea pigs: effects of age, passive immuni- accounted for by the immunization procedure, zation, and hydrocortisone. Infect. Immun. 16:69-74. 11. WalZ, M. A., H. Yamamoto, and A. L. Notkins. 1976. which avoided the establishment of latent infec- Immunologic response restricts number of cells in sen- tions by intraperitoneal administration of the sory ganglia infected with herpes simplex virus. Nature virus. In our present and previous experiments (London) 264:554-556. 12. Wohlenberg, C. R., M. A. Walz, and A. L. Notkins. (4) a total of 65 mice surviving the primary 1976. Efficacy of phosphonoacetic acid on herpes sim- infection in the lumbosacral region were reino- plex virus infection of sensory ganglia. Infect. Immun. culated in the orofacial area. In 46 mice without 13:1519-1521.
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