J. gen. Virol. U976), 3 x, 75-80
75
Printed in Great Britain
Latent Herpetic Infections Following Experimental Viraemia
By M A R G E R Y L. C O O K AND J. G. S T E V E N S
Reed Neurological Research Center and Department of Microbiology and Immunology,
UCLA School of Medicine, Los Angeles, California 9oo24, U.S.A.
(Accepted 26 November 2975)
SUMMARY
The spectrum of tissues harbouring latent herpes simplex virus following
intravenous inoculation of mice was defined by in vitro co-cultivation techniques.
The virus could be detected in central and peripheral nervous systems (including
adrenal medulla), but could not be found in any non-neural tissues. Spinal
ganglia were the organs most commonly involved. The relationship of these
findings to the natural history of herpetic infections is discussed.
INTRODUCTION
Following peripheral inoculation in experimental animals, herpes simplex virus (HSV)
moves in sensory nerves to corresponding ganglia and then to the central nervous system
(Cook & Stevens, 1973). In both peripheral and central nervous systems (CNS) the resultant
acute disease (which may be asymptomatic) is followed by a long standing latent infection
from which infectious virus can later be reactivated by in vitro cultivation of the involved
tissue (Stevens & Cook, 2971; Knotts, Cook & Stevens, 2973). It now seems quite likely
that these phenomena also obtain in man (cf. Nahmias & Roizman, I973), although direct
evidence for latent infection in the CNS has yet to be obtained.
Haematogenous spread of herpes simplex infections also occurs, and many organs, including the nervous system, are 'seeded' by this route (Johnson & Mims, 2968; Nahmias &
Roizman, 2973). Although acute cytolytic infections in these tissues may follow the viraemia,
the possibility that latent infections are subsequently established has not been investigated.
In this paper we show that, in the mouse, latent infections of both peripheral and central
nervous systems do follow a viraemia and that sensory ganglia are commonly involved. In
this model, latent infections of haematogenous tissues and various other organs were not
detected.
METHODS
Outbread Swiss mice 4 or 8 weeks of age were inoculated in a tail vein with 3 x Io 3 to
3 x IO plaque forming units (p.f.u.) of HSV. The virus employed (type I, McIntyre strain)
was grown and assayed as previously described (Cook & Stevens, 1973).
After an interval of 28 to IO9 days, mice were sacrificed and spinal ganglia, spinal cords,
trigeminal ganglia, adrenal glands, lymph nodes, spleen, bone marrow, liver, lung, and
kidney were cultured by methods which we have previously employed for detection of both
overt and latent virus infections (Knotts, Cook & Stevens, 2974). First, the brain was
removed from each mouse and the separated cerebellum and brain stem together made up
the sample designated posterior brain. The remainder of the brain (anterior brain) was cut
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76
M. L. C O O K
AND
J. G. S T E V E N S
transversely and the left hemisphere was used. Next, the trigeminal ganglia were removed
and processed together. After the entire vertebral column had been cut longitudinally with
iris scissors, the exposed spinal cord was removed intact. Lumbar and sacral spinal ganglia
on both sides were then dissected out and the I4 to I8 ganglia recovered were pooled and
became the posterior ganglia sample. Anterior ganglia consisted of both cervical and thoracic
spinal ganglia (25 to 3o ganglia). Bone marrow was expelled from the tibia and femur of
one hind leg of each mouse using a 27 gauge needle and syringe filled with balanced salt
solution. The remainder of the specimens consisted of adrenal glands, pooled mesenteric
and sciatic lymph nodes and representative portions ( ~ 75 mg) of spleen, liver, lungs, and
kidney all processed individually. In all but the specimens of spinal ganglia and bone marrow,
tissues were chopped into pieces about I mm in diam. and then co-cultivated with monolayers of R K I3 cells. Spinal ganglia and bone marrow specimens were co-cultivated without
further treatment. The R K I3 cell monolayers were scored for virus-specific c.p.e, and supernatant fluids for positive cultures were frozen at - 7 o °C for later viral identification. Cultures not demonstrating virus-specific c.p.e, by 28 days of co-cultivation were discarded as
negative. In this regard, it is important to note that all positive cultures were detected by the
23rd day of co-cultivation, and that 95 % of the positives were detected by the I3th day.
Nine viral isolates selected at random from the cultures demonstrating typical herpetic
c.p.e, were identified as HSV by neutralization tests with HSV specific antisera (Knotts et al.
I973). Finally, the methods used for ultrastructural studies have been detailed elsewhere
(Cook & Stevens, I97o).
RESULTS
Clinical course of disease
The accumulated results of seven experiments (a total of I72 mice inoculated) served as
a basis for description of the clinical illness. Thus, after intravenous inoculation of HSV,
signs of disease were detected in 5o to 75 ~ of mice on days 5, 6 and 7; the remainder remained free of disease. Those animals presenting clinically apparent disease had difficulty
in locomotion which often progressed to a flaccid paralysis involving one or both rear legs.
Ten to 70 ~ of these mice later died with encephalitis. Most paralysed mice which did not
develop encephalitis recovered clinically in 2 to 4 weeks; a minority were left with minor
rear leg weakness.
Tissues harbouring latent infections after intravenous inoculation of virus
The pooled results of 4 separate experiments in which individual tissues from surviving
mice were assayed for latent HSV are presented in Table I. Of particular significance are
the following findings: first, latent infections were limited to the nervous system and one
adrenal gland. Secondly, more than 9o ~ of the animals harboured latent virus in one or
more of the tissues examined. Lastly, dorsal root ganglia were the sites most commonly
involved (83 ~ of mice compared with 25 ~ in CNS).
Effect of tail amputation on establishment of latent infections
Although the virus was injected intravenously, it could reasonably be argued that
leakage at the injection site resulted in infection of local nerve endings, centripetal passage
of virus through nerves, and subsequent establishment of latent infections in the nervous
system. To investigate this possibility the tails of infected mice were amputated 2"5 cm
proximal to the injection site immediately following injection. The results from this study
(Table 2) are comparable to those reported above (82 ~o of animals possess latent infections,
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Latent herpetic infections after viraemia
T a b l e I. I n v i t r o
Animal
number
Virus
inoculated
(p.f.u.)
77
reactivation of latent herpes simplex virus from various murine tissues
following intravenous :noculation
Time
between
Age at infection
time of
and
infection sacrifice
(weeks)
(days)t
Tissues processed for detection of
latent infections, and results*
2,
Anterior Posterior
brain
brain
Spinal
cord
Adrenal
gland
I
3000
4
2
3 000
4
10 9
3
3 ooo
4
i o9
+
+
4
5
6
7
8
9
Io
80o0
8ooo
8ooo
3oooo
4
4
4
8
8
8
8
8
8
56
56
56
27
27
27
35
35
35
+
+
+
+
+
+
+
ii
I2
30000
3OOOO
30000
30 ooo
30000
IO9
Anterior Posterior
spinal
spinal
ganglia ganglia
+
* No virus could be detected in trigeminal ganglia, bone marrow, lymph nodes, spleen, kidney, lung, or
liver processed by the same method. Further details concerning methods are given in the text.
t All mice had demonstrated neurological signs within 7 days after infection and numbers 7 to IO were
left with a residual posterior paralysis apparent at the time of sacrifice.
T a b l e 2. I n v i t r o
Animal
number
reactivation of latent herpes simplex virus from various murine tissues
following intravenous inoculation and tail amputation
Time
between
Age at infection
Virus
time of
and
inoculated infection sacrifice
(p.f.u.)
(weeks)
(days)
I*
2
20000
20000
4
4
18
3
4
5
20 ooo
4
20000
2oooo
4
4
6
2o ooo
4
19
7
20 ooo
8
2I
8
20 ooo
8
zi
9
IO
II
20 000
20000
20000
8
8
8
21
22
22
Tissues processed for detection of latent infections, and results
r
Anterior Posterior
brain
brain
+
Spinal
cord
Adrenal
gland
Anterior Posterior
spinal
spinal
ganglia ganglia
+
+
+
i8
]8
19
19
+
+
+
+
+
+
+
+
+
* Mouse number one demonstrated persistent posterior paralysis beginning on the 6th day after infection.
All other mice remained clinically normal. Further details concerning methods are given in the text.
82 ~ p o s i t i v e in g a n g l i a a n d I8 ~ooin C N S ) . I n a d d i t i o n , t w o m o r e a d r e n a l glands w e r e f o u n d
to h a r b o u r l a t e n t H S V .
F i n a l l y , e m p l o y i n g t e c h n i q u e s w h i c h we h a v e d e s c r i b e d earlier ( K n o t t s et al. 1974)
a t t e m p t s w e r e m a d e to isolate virus d i r e c t l y f r o m brains, spinal cords, spinal g a n g l i a a n d
a d r e n a l glands f r o m 6 a d d i t i o n a l mice selected at r a n d o m f r o m these t w o g r o u p s . N o virus
c o u l d be detected.
(i
VIR 31
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78
M.L. COOK AND J. G. STEVENS
Fig. L Photomicrograph of an adrenal gland from a mouse previously infected intravenously with
HSV. This specimen was co-cultivated with RKI3 cells for IO days before it was processed for
electron microscopy. Several cells shown (arrowed) in the adrenal medulla exhibit margination of
chromatin and central nuclear clearing. This appearance is typical of cells found to be infected
with HSV in specimens prepared by these methods. Such cells were absent in the adrenal cortex (C).
Fig. 2. An electron micrograph of a herpesvirus-infected medullary cell from the specimen seen
in Fig. I. Viral nucleocapsids (shown arrowed) and many granules typical of those present ia
chromaffin cells are seen in the cytoplasm. Four immature viral forms can be seen in the nucleus
and three virions are outside the cell.
Morphological characteristics of the reactivated infection in adrenal glands
I n a d r e n a l specimens in which active herpes virus infection could be detected, the m e d u l l a
a p p e a r e d to be selectively involved. A n example is presented in Fig. I where several cells in
the m e d u l l a r y p o r t i o n o f the g l a n d d e m o n s t r a t e d c.p.e, typical o f herpes virus infection.
This specimen was processed after ro days o f co-cultivation a n d at the time the first c.p.e.
a p p e a r e d in the R K ~3 cell m o n o l a y e r . A n electron m i c r o g r a p h o f one o f the cells f r o m this
specimen is presented in Fig. 2. Here, chromaffin granules (typical o f m e d u l l a r y cells) a n d
various structural forms characteristic o f herpes viruses can be seen. O t h e r infected cells in
this specimen a p p e a r e d to be either ganglion or s u p p o r t i n g cells. N o cells in the adrenal
cortex were seen to be infected at this time.
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Latent herpetic infections after viraemia
79
DISCUSSION
The results presented here indicate that, following intravenous infection, HSV selectively
establishes latent infections in neural tissue, particularly in sensory ganglia. That the blood
stream is in fact involved in seeding these tissues was shown conclusively when inoculation
was followed by tail amputation (Table 2). However, it should be noted that these experiments do not prove that the nervous tissues were seeded directly by virus in the bloodstream;
they could have become infected indirectly by virus travelling in nerves associated either
with vessels or other organs originally seeded by the viraemia. It can also be tentatively concluded that the virus does not establish latent infections in any of the extraneural tissues
examined. Here, however, two obvious reservations must be appended. First, the amount
of virus injected may have been insufficient (even though IO to 70 ~o of mice died with acute
encephalitis and all mice presented in Table I had been paralysed). Secondly, methods
employed for reactivation of virus in nervous tissues may be inadequate to reactivate the
agent from other tissues. This reservation is somewhat blunted, however, since similar
methods have been used to reactivate other herpes viruses from lymphoid tissues (cf. Symposium on Herpes Virus and Cervical Cancer, 1973).
Our recovery of latent virus from the brains of a minority of mice following intravenous
infection permits a reasonable explanation for the origin of virus responsible for sporadic
herpetic encephalitis in man. Thus, at least some of these cases may well be initiated by a
reactivated virus originally seeded by viraemia and harboured in the CNS of a few individuals. In another extension to man, the results with sensory ganglia might be considered
in relation to a controversy involving the pathogenesis of Herpes zoster. It is now generally
assumed that sensory ganglia are the source of virus in zoster, but it is not clear whether
ganglia are originally seeded via associated nerves, by haematogenous routes, or by both
routes. Although our data could be viewed as evidence for direct haematogenous seeding,
here again the possibility of neural spread is not ruled out.
Finally, the demonstration that latent infection can be established in the adrenal gland,
and that it is apparently restricted to the medulla is significant. The adrenal medulla is of
neural origin and contains sympathetic ganglion cells which are infected during viral reactivation. This result, those presented above, and our earlier studies of selective latent infections in sensory neurons following inoculation of the skin (Stevens, I975) lead to the working
hypothesis that latent HSV infections are limited to nervous tissues, and, more specifically,
to the neurons in these tissues. The biochemical basis of such a restricted and unique viruscell interaction is, at present, not understood.
This investigation was supported by grants from the National Institutes of Health,
United States Public Health Service (Numbers A[-o6246 and NS-o871 I).
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M.L. COOK
AND
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