Latent Herpes Simplex Virus Infections in Sensory Ganglia of by liaoqinmei


									                                       Latent Herpes Simplex Virus
                                       Infections in Sensory Ganglia
                                       of Hairless Mice Prevented by
                                       Richard J. Klein, Alvin E. Friedman-Kien
                                       and Eugene DeStefano
                                       Antimicrob. Agents Chemother. 1979,
                                       15(5):723. DOI: 10.1128/AAC.15.5.723.

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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 1979, p. 723-729                                                   Vol. 15, No. 5

 Latent Herpes Simplex Virus Infections in Sensory Ganglia of
         Hairless Mice Prevented by Acycloguanosinet
     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

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            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
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

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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
       3                                             flofl SUrViVg days or initiated after a delay of 24 h protected
 0                                                St
                                                  \        t       only 30% of the mice (Table 3). The difference
                                                                   was statistically significant (P < 0.04). Latent
                                                                   HSV infections in the trigeminal ganglia were
      2                                                            observed in 70% of the mice treated with 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

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       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
                                              A                    weeks after inoculation, by heart puncture. Se-

                                       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
                                               all surviving       human foreskin cells as previously described (6).
o            -                                          V             Despite a relatively wide range of antibody
                  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
                                                                   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-
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
  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

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                              24                       0.65                 0/10                2/10
                              48                       1.45                 0/10                5/10
  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
        Treatment        Treatment schedule         Avg lesion scorea     Mortality rate        fection
  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
  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
     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
                                                                         glia. Only 1 mouse was found to have latent
              O   obsent                                         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.

              3hpi 3h pi 24hpi 48hpi 3hpi 3hpi 24hpi
              S days 3days 5doys Sdays 15doys 3days Sdoys
                                                              3 h pti
                                                                           Our results indicate that HSV-induced skin

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                     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
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.
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.

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    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 +
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

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 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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