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J Virol, August 1998, p. 6888-6892, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The Probability of In Vivo Reactivation of Herpes
Simplex Virus Type 1 Increases with the Number of Latently Infected
Neurons in the Ganglia
N. M.
Sawtell*
Division of Infectious Diseases, Children's
Hospital Medical Center, Cincinnati, Ohio 45229-3039
Received 2 January 1998/Accepted 30 April 1998
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ABSTRACT |
The purpose of this study was to define the relationship
between herpes simplex virus (HSV) latency and in vivo ganglionic reactivation. Groups of mice with numbers of latently infected neurons
ranging from 1.9 to 24% were generated by varying the input titer of
wild-type HSV type 1 strain 17syn+. Reactivation of the virus in mice
from each group was induced by hyperthermic stress. The number of
animals that exhibited virus reactivation was positively correlated
with the number of latently infected neurons in the ganglia over the
entire range examined (r = 0.9852, P < 0.0001 [Pearson correlation]).
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TEXT |
Herpes simplex virus (HSV) is a
neurotrophic pathogen of humans that establishes latent infections in
the sensory ganglia innervating the site of primary disease. The latent
virus periodically reactivates, producing infectious virus which can
result in recurrent surface lesions (for reviews, see references
23 and 35). The frequency with
which infected individuals experience clinically manifested
reactivation of HSV is quite variable, ranging from 0 to 12 or more episodes per year (30; see reference
35 for a review). Why this is the case is not
understood, but the number of latent sites in the ganglia is one factor
that may be important.
The correlation between the amount of HSV latency and the frequency of
reactivation both in vitro and in vivo has been examined by a number of
investigators. In these studies, latency was quantified on the basis of
(i) the total amount of viral DNA in latently infected ganglia (1,
2, 4, 8, 10, 13-16, 34), (ii) the number of latency-associated
transcript (LAT) RNA-positive or LAT promoter reporter-positive neurons
(5, 7, 26), or (iii) the number of neurons containing the
viral genome as determined by PCR-based approaches (16, 33).
Steiner et al. reported a direct correlation between input PFU and
cocultivation reactivation of HSV in the trigeminal ganglia (TG) of
mice inoculated with a VP16-negative mutant (32). In all but
one of these reports, mutant virus and/or wild-type strains differing
in their ability to reactivate were compared. Leib et al. examined the
correlation between input titers of HSV strain KOS, the amount of viral
DNA in the latently infected ganglia, and cocultivation reactivation (15). A reduction of cocultivation reactivation was observed only with very low input titers. The level of establishment could not
be quantified in these ganglia because of the insensitivity of the slot
blot hybridization method employed (15).
These studies have extended our understanding of the link between acute
infection, the establishment of latency, and subsequent reactivation.
However, a basic issue remains unresolved. Does the number of latently
infected neurons influence the in vivo HSV reactivation potential of
ganglia infected with a given virus strain? If so, what is the nature
of the relationship? Recent estimates made by using PCR-based assays
for the viral genome indicate that as many as 10 to 30% of the neurons
can be latently infected, a number significantly larger than previously
concluded on the basis of in situ detection of LAT RNA-expressing sites (16, 18, 21, 22, 24). Delineating the relationship between this number and reactivation would provide clues to the mechanism of
reactivation and aid in establishing clinical treatment goals. In this
study, the ability of HSV type 1 (HSV-1) strain 17syn+ to reactivate
from ganglia containing different numbers of latently infected neurons
was determined. A recently developed method, contextual analysis of DNA
(CXA-D), was used to quantify virus latency at the single-cell level.
Using this assay, the percentage of ganglionic neurons containing viral
DNA can be determined (24).
Correlation among input titer, PIN, and in vivo reactivation.
Groups of male Swiss Webster mice (18 to 20 g) obtained from
Harlan Laboratories (Indianapolis, Ind.) were infected on scarified corneas with input titers of wild-type strain 17syn+ (obtained from J. Subak-Sharpe of the Medical Research Council Virology Unit in Glasgow,
Scotland) ranging from ~5 × 102 to ~5 × 105 PFU. Preliminary experiments demonstrated that this
3-log span of inoculum titers resulted in significant differences in
the numbers of latently infected neurons in the ganglia. Higher input titers resulted in unacceptable levels of mortality, and lower input
titers increased the probability that the mice would not be infected.
At >30 days postinoculation, six ganglia from each group were
processed and then analyzed by CXA-D (24). In brief, perfusion-fixed trigeminal ganglia were removed and dissociated into
single-cell suspensions, and enriched neuron populations were obtained
by using Percoll (Pharmacia) gradients. The percentage of
infected neurons (PIN) in the latently infected ganglia was determined
by a single-neuron PCR assay as described previously (24).
Consistent with our previous report (24), reducing the input
titer resulted in ganglia containing fewer latently infected neurons
(Table 1). Input titers of
~105 PFU resulted in >20% of the ganglionic neurons
harboring HSV DNA, while only ~2% of the neurons were positive when
the input titer was reduced to 500 PFU.
Groups of mice, each inoculated with a different input titer, were
subjected to hyperthermic stress (HS) to induce HSV reactivation
as
described previously (
25). At 22 h posttreatment, the
time
at which peak amounts of infectious virus are detected in the
ganglia (
25), the trigeminal ganglia were removed,
homogenized,
centrifuged to remove cellular debris, and plated on
rabbit skin
cell (RSC) monolayers. The plates were monitored for 5 days, but
all primary plaques became evident within 24 to 36 h
postplating.
There was a positive correlation between the input titer and the number
of mice in the group that exhibited HSV reactivation
following HS (Fig.
1). Seventy and 60% of the mice in the
two
high-input-titer groups demonstrated HSV reactivation. These groups
contained the largest number of HSV-positive neurons, 24 and 20%,
respectively. With an intermediate input titer, 11.5% of the neurons
were latently infected, and 30% of the mice in this group showed
viral
reactivation. At the lowest input titer, only 1.9% of the
neurons were
latently infected, and 5% of the mice from this group
exhibited HSV
reactivation.
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FIG. 1.
The percentage of mice undergoing viral reactivation
following HS. Groups of latently infected mice inoculated with the
input titers indicated were subjected to HS. Shown are the numbers of
mice exhibiting viral reactivation over the total number treated. There
was no difference between the two groups receiving input titers of
>105 PFU (two-sided P = 0.7, Fisher's
exact test). For statistical analysis, these two groups were combined
and compared to the groups receiving either 2 or 3 logs less input
virus. The differences were significant (P = 0.04 and
P < 0.0001, respectively, Fisher's exact test).
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Consistent with previous reactivation experiments, only a few PFU were
recovered from a given reactivation event, and this
did not vary among
the groups (data not shown). The importance
of the sensitivity of the
reactivation assay for the interpretation
of these results warranted
examination of this issue. The sensitivity
of detection of infectious
virus in this assay had been previously
approximated by a simple
spiking experiment (
25). Repeating
this experiment under the
laboratory conditions used in the present
study demonstrated that when
20 PFU was added to uninfected ganglia
and the ganglia were then
homogenized and plated on RSC monolayers,
an average of 6 PFU was
detected. This experiment, however, did
not evaluate the efficiency of
virus recovery when the ganglia
were latently infected or in the
process of HSV reactivation.
To examine this, a human cytomegalovirus
immediate-early promoter-
lacZ reporter mutant, KZ, kindly
provided by J. Mester, was utilized
to spike latently infected ganglia
removed pre-HS or at 22 h post-HS.
The construction and
characterization of this mutant have been
previously described
(
20). It was now possible to distinguish
input virus from
latent virus reactivating in the ganglia on the
basis of
lacZ expression, detected as blue plaques as described
elsewhere (
20). A comparison of the number of blue plaques
recovered
on RSC monolayers when virus was (i) plated in the absence of
ganglion homogenate, (ii) homogenized with uninfected ganglia
(pre-HS
or 22 h post-HS), and (iii) homogenized with latently
infected
ganglia (pre-HS or 22 h post-HS) was performed. The assay
for each
group was run in triplicate. The results of this experiment
were
consistent with those of the previous one in that on average,
16 blue
plaques were evident when 50 PFU was added to uninfected
ganglia. HS
did not alter the recovery. When 50 PFU was added
to latently infected
ganglia and those ganglia were then homogenized,
10 plaques were
observed on average. The reason for this approximately
twofold
reduction in sensitivity was not examined, but it may
represent the
presence of immune factors in the latently infected
ganglia
(
29). Interestingly, there was no difference in the
efficiencies of exogenous virus recovery from latently infected
ganglia
pre- and post-HS. This experiment indicates that the assay
for the
detection of infectious virus in a reactivating ganglia
is quite
sensitive, but depending on the amount of virus in the
ganglia at
22 h post-HS, some reactivation events would go undetected.
In
addition, it provides further confirmation that the striking
difference
in the levels of virus recoverable from the ganglia
during acute
infection (10
4 PFU on day 4 postinoculation with
17syn+) and during reactivation
(1 to 20 PFU) reflects the levels being
produced and not merely
immune factor interference with
detection.
ACV treatment reduces the number of latent infections and
subsequent in vivo reactivation.
We had previously shown that
acyclovir (ACV) administered during acute infection resulted in a
reduction of the number of latently infected neurons in the ganglia of
treated mice (24). Therefore, an additional experiment was
performed to test whether controlling the number of latently infected
neurons in this manner has a similar impact on reactivation. This would
test the hypothesis that the viral reactivation frequency is determined
by the input titer and not by events, such as replication, occurring
subsequent to inoculation. Male Swiss Webster mice (18 to 20 g)
were inoculated on scarified corneas with 2.6 × 105
PFU of wild-type HSV-1 strain 17syn+. A 50-mg/kg dose of ACV (Glaxo
Wellcome) was administered to each mouse intraperitoneally three times
per day beginning either at the time of inoculation or following a
delay of 36 h and continuing through day 7 postinoculation. Control mice received saline alone. As predicted, acute virus replication was markedly reduced in the eyes and TG of ACV-treated mice, confirming the efficacy of treatment (data not shown). At >30
days postinoculation, the percentage of neurons latently infected in
each group was determined by CXA-D (24). For each group, neurons harvested from six ganglia were pooled and analyzed. ACV treatment dramatically reduced the number of latently infected neurons
in the ganglia, a finding consistent with our previous report (Fig.
2) (24). Even when ACV
treatment was delayed for 36 h, the number of latently infected
neurons was reduced >10-fold compared to the number in the ganglia of
sham-treated control mice. As described above, HSV reactivation in mice
from each of these groups was induced by HS (25). The
results showed a clear correlation between the number of latently
infected neurons in the ganglia and the number of mice in which HSV
reactivation occurred following HS (Fig. 2). In addition, the
possibility that the reactivation frequency was determined directly by
the input titer was eliminated.
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FIG. 2.
The percentage of neurons latently infected and the
percentage of mice that exhibited HSV reactivation following HS in ACV-
and sham-treated groups. The percentage of latently infected neurons in
ganglia of sham-treated mice and mice which had been treated with ACV
from the time of inoculation (ACV0) or from 36 h
postinoculation (ACV36) through day 7 postinoculation was
determined by CXA-D. Viral reactivation was induced in mice from each
of these groups. The differences between the number of latently
infected neurons and the number of mice exhibiting reactivation in the
sham- and ACV-treated groups were both significant (two-tailed
P < 0.0001 for both ACV treatment groups, Fisher's
exact test).
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Variability in PIN among animals.
In the preceding
experiments, the number of latently infected neurons in each of the
groups of mice was determined by analyzing a pool of six ganglia from
mice representing that group. Thus, there was a possibility that the
reactivation data at the lower input titers reflected the reactivation
of virus in one animal in the group which had a very high percentage of
latently infected neurons. It was therefore important to gain insight
into the degree of variability among the individual mice in each
input-titer group. To do this, groups of mice were inoculated, via
corneal scarification, with either 500, 5,000, or 500,000 PFU of HSV.
At >30 days postinoculation, mice were perfusion fixed and ganglia
from individual mice were analyzed by CXA-D to determine the number of
latently infected neurons. Eight individual mice in the 500-PFU group,
six in the 5,000-PFU group, and five in the 500,000-PFU group were
analyzed. All of the inoculated mice examined were found to contain
latently infected neurons. The PIN was most variable in the 500-PFU
input-titer group, ranging from 0.16 to 4.6% (mean, 1.5%), while
those of the 5,000- and 500,000-PFU groups ranged from 5 to 18% (mean, 10%) and 24 to 29% (mean, 27%), respectively. The average PIN in the
input-titer groups in this experiment were very similar to those
obtained in the first experiment, 1.9, 11.5, and 24%, respectively. As
presented below, seven mice in the 500-PFU group were also analyzed by
whole-ganglion quantitative PCR (QPCR), and none of the TG contained
high levels of HSV DNA. Thus, in a total of 15 individual mice and a
pool of 3 mice of the lowest-input-titer group, no outliers were
detected. It therefore seems unlikely that HSV reactivation in the
500-PFU group was due to the presence of a single animal with a very
large number of latently infected neurons.
Total-ganglion QPCR.
To compare the efficiency of detection of
low levels of latency by total-ganglion QPCR with that of single-neuron
PCR, DNA was prepared from TG of seven mice belonging to the 500-PFU
inoculation group (described above) and was analyzed by the method of
Katz et al. (11). HSV DNA was detected in only three of the
seven ganglion pairs; one contained an estimated 26,500 viral genomes, and the other two had levels too low to accurately quantify. The standards in this assay demonstrated that in the background of 100 ng
of mouse DNA, 50 HSV genomes were detectable but 5 HSV genomes were
not. This meant that ganglion pairs containing significantly fewer than
50 HSV genomes per 100 ng of mouse ganglion DNA could appear to be
negative. Thus, in line with the sensitivity of the QPCR assay, HSV was
detected in those ganglia containing the largest number of latently
infected neurons but not in those containing the fewest. Two previous
studies have demonstrated that estimates of total HSV DNA obtained by
whole-ganglion QPCR are consistent with those obtained by CXA-D
single-neuron PCR (24, 28).
The total number of latently infected neurons per TG pair was
calculated for each group by multiplying the percentage of neurons
latently infected by 40,000 (total neurons per TG pair) (
3,
24) (Table
1). This number was then used to determine the
covariation
or correlation between a detectable reactivation event and
the
numbers of latently infected neurons present in the groups of
mice
infected with different HSV titers. In the group of mice
receiving the
smallest amount of virus, a detectable reactivation
event (i.e., a
positive animal) was detected once in every 15,200
latently infected
neurons. In those animals that received the
most virus, this number was
once in every 13,700 latently infected
neurons. It should be emphasized
that this represents the minimum
number of reactivation events which
occurred in vivo. An additional
assumption is that all latently
infected neurons have an equal
probability of HSV reactivation. The
relationship between the
number of latently infected neurons in the
ganglia and in vivo
reactivation frequency is shown graphically in Fig.
3. For this
analysis, data from both the
input-titer and the ACV experiments
were included. Although there is a
strong correlation between
the frequency of HSV reactivation and the
number of latently infected
neurons (
r = 0.9852,
P < 0.0001), this does not indicate that
there is a
causal link between these two parameters. Unfortunately,
there is
currently no method for quantification of the number
of latently
infected neurons and assessment of viral reactivation
in the same
ganglia. These data do suggest, however, that large
numbers of latently
infected neurons in the ganglia do not necessarily
lead to detectable
HSV reactivation in all animals and, further,
that some animals with
relatively few latently infected neurons
undergo viral reactivation.
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FIG. 3.
Plot of the number of latently infected neurons per TG
pair versus the percentage of mice exhibiting reactivation. Data from
both the input-titer experiment and the ACV experiment were included.
The number of latently infected neurons per TG pair was determined from
the PIN for each input titer group. There is a significant correlation
between these two variables (r = 0.9852, P < 0.0001 [Pearson correlation test]).
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Human HSV infection has not been explored at this level of detail.
However, it is reasonable to assume that, as in the mouse,
the number
of latently infected neurons resulting from primary
infection in the
human could be a significant determinant of the
frequency of HSV
reactivation. In humans, the frequency of reactivation
events resulting
in recurrent disease is related to the severity
of primary infection
(for a review, see reference
35). This
is consistent
with the impact of the input titer on the number
of latently infected
neurons reported here as well as the reduction
in the number of latent
sites in ganglia of mice treated with
ACV during acute infection. Thus,
while the complete prevention
of latent infections may not be possible,
a practical and achievable
clinical goal would be minimization of the
number of neurons in
which establishment of latency occurs. Empirical
evidence that
transmission of an alphaherpesvirus can be reduced in a
population
through the control of viral replication and shedding is
provided
by the success of immunization strategies against pseudorabies
virus in pig herds (
19,
31).
There are several lines of evidence which suggest that the HSV in the
vast majority of latently infected neurons does not
reactivate during
any given reactivation event. The most compelling
of these is the fact
that HSV can reactivate many times over the
lifetime of the human host.
If one accepts the theory that viral
DNA replication is not compatible
with cell survival (
23) and
the evidence that the neuron in
which the virus reactivates does
not survive (
25,
29), then
the conclusion that the virus reactivates
in very few neurons per
episode is reasonable. Animal model studies
support this in that (i)
the latent-DNA reservoir appears to be
stable over long periods of time
(
9) and (ii) examination of
ganglia in which HSV is
reactivating has revealed that very few
neurons express lytic viral
proteins or ICP0 RNA (
5,
6,
17,
25,
29). We have found that
neurons expressing viral
lytic proteins post-HS are rare even in
ganglia in which >20%
of the neurons contain the latent viral genome
(
27). If many
neurons produced very low (undetectable)
levels of viral proteins
and a few infectious virions post-HS, one
would predict an increased
recovery of virus from mice in which many
(>20%) of the neurons
were latently infected. However, the amount of
virus recovered
per reactivation did not increase with increasing
numbers of latently
infected neurons in the ganglia.
PCR-amplifiable segments of RNA related to lytic genes have been
detected in latently infected ganglia in which infectious
virus cannot
be detected, suggesting that there are more neurons
in which the latent
viral transcriptional program is exited than
there are neurons that
proceed to detectable infectious virus
production (
12).
Nonetheless, the findings presented here indicate
that neuronal and/or
viral mechanisms serve to maintain the viral
latent state in the vast
majority of infected neurons. The viral
genome copy number in
individual latently infected neurons has
been hypothesized to play a
critical role in reactivation (
23).
We have found that while
the majority of neurons latently infected
with HSV-1 strain 17syn+
contain between 10 and 100 viral genomes
(
24), extremely
rare neurons contain copy numbers in the range
of 5,000 to 10,000 (
27). It may be that only these very-high-copy-number
neurons are reactivation competent in response to HS induction
in vivo.
The relationship between these rare neurons and reactivation
is
currently under investigation.
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ACKNOWLEDGMENTS |
I thank R. L. Thompson for helpful discussion and C. S. Tansky for expert technical assistance.
This work was supported by Public Health Service grants AI32121 (from
the National Institutes of Allergy and Infectious Diseases) and NS25879
(from the National Institutes of Neurological Communicative Disorders
and Stroke) and CHMCC Trustee grant 31-358-639.
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FOOTNOTES |
*
Mailing address: Division of Infectious Diseases,
Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH
45229-3039. Phone: (513) 636-7880. Fax: (513) 636-7655. E-mail:
Sawtn0{at}CHMCC.org.
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