Download Experimental Infection of Inbred Mice with Herpes Simplex Virus. V

J. gen. Virol. (1982), 63, 315 323. Printed in Great Britain
315
Key words: herpes simplex virus/virulence and pathogenesis/interferon/immunity
Experimental Infection of Inbred Mice with Herpes Simplex Virus. V.
Investigations with a Virus Strain Non-lethal after Peripheral Infection
By G. K U M E L , * H. K I R C H N E R , R. Z A W A T Z K Y , H. E N G L E R ,
C. H. S C H R O D E R AND H. C. K A E R N E R
Institute for Virus Research, German Cancer Research Centre, Heidelberg,
Federal Republic of Germany
(Accepted 7 July 1982)
SUMMARY
The herpes simplex virus type I(HSV-1) strain A N G , unlike the majority of HSV-1
isolates, does not cause lethal encephalitis in various inbred mouse strains, when
applied in doses up to 2 x 107 plaque-forming units intraperitoneally, intravenously,
intravaginally, orally, or via the foot pads. We studied and compared the progress of
infection by this non-lethal strain and by a lethal HSV-1 strain, and the components of
the host defence mechanism involved. If injected intracerebrally, HSV-1 A N G
replicated efficiently in mouse brain cells and led to encephalitis. Upon systemic or
peripheral infection, it replicated in several mouse organs with a virulence similar to
lethal HSV-1 isolates. It was clear that transport of HSV-1 A N G to the central nervous
system (CNS) or replication in CNS tissue is efficiently restricted after peripheral
infection. Conceivably, infection with lethal HSV-1 strains proceeds in two distinct
steps, virus replication at the site of infection and in the spleen and, secondly, transport
to CNS tissue and propagation in CNS cells. This second step is apparently blocked in
infections of DBA/2 mice by HSV-1 A N G and can thus be studied separately. The
blocking mechanism was not a function of interferon induction or sensitivity, nor was it
due to an enhanced N K cell activation. Experiments with silica-treated mice, and with
homozygous nude mice, which lacked T-lymphocytes, suggested that the observed
restriction in virus transfer is independent of T-cell and macrophage functions. Yet,
newborn mice were fully susceptible to intraperitoneal infection with HSV-1 A N G ,
suggesting that age-dependent defence mechanisms, the nature of which needs to be
further examined, are of relevance in the restriction of peripheral infection by HSV-1
A N G in adult mice.
INTRODUCTION
Herpes simplex virus type 1 (HSV-1) is a neurotropic human virus of considerable clinical
importance, particularly in immunoincompetent or immunosuppressed individuals (Nahmias et
al., 1981). With the aim of better understanding the pathogenesis of HSV and the host defence
mechanisms interacting with the infection, model systems have been established in a number of
laboratories (including our own) using HSV infection of mice (e.g. Andervont, 1927; Zisman et
al., 1970; Stevens & Cook, 1971 ; Lopez, 1975; Kirchner et al., 1978). It is known that different
strains of HSV-1, even primary clinical isolates, differ considerably in their lethality for mice
(Hill et aL, 1975). Thus genetic differences between HSV-1 virus strains may be responsible for
differences in pathogenicity.
Most studies concerned with the mechanism of pathogenicity of HSV in mice have been
performed using virus strains that cause lethal encephalitis in susceptible inbred mice at
moderate virus doses. It is of interest to compare the spread of virus infection following
peripheral infection with HSV strains of different pathogenicity patterns. Consequently, this
study is concerned with the characterization of the infection of inbred mice with HSV-I A N G , a
non-lethal HSV strain.
0022-1317/82/0000-5175 $02.00
© 1982 SGM
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G. KUMEL AND OTHERS
In the past few years, the molecular genetics o f HSV-I A N G have been extensively studied in
our laboratories (Schr6der et al., 1975/76; K a e r n e r et al., 1981; Kiimel et al., 1981, 1982).
Recently, we have found that HSV-I A N G is non-lethal for mice after intraperitoneal (i.p.)
infection (Schr6der et al., 1981). This was in marked contrast to HSV-1 W A L which has been
commonly used in studies in our laboratory and which kills certain strains of inbred mice after
i.p. injection of low doses of infectious particles (Kirchner et al., 1978).
In this study, we analyse the spread of HSV-1 W A L and HSV-1 A N G in different tissues at
various times after i.p. inoculation and attempt to define the host mechanisms which mediate
the observed difference in behaviour of the two virus isolates.
METHODS
Cells and virus. HSV-I strains were routinely passaged at low multiplicity on African green monkey kidney cells
(RC-37 Rita; Italdiagnostics, Rome, Italy) as described earlier (Schr6der et al., 1975/76). Cells were grown in
Eagle's minimal essential medium (MEM) containing 7~ foetal calf serum. Plaque-forming virus was assayed as
described by Russel (1962). Origin and characterization of the HSV-I strains ANG and WAL have been described
earlier (Darai & Munk, 1976; Schr6der et al., 1975/76, 1981).
Mice. The animal model used has been described in detail (Zawatzky et al., 1981). Inbred mouse strains,
C57BL/6 nu/nu mice and their heterozygous litter-mates were purchased from GI. Bomholtgard Ltd. (Ry,
Denmark) and from Deutsche Gesellschaft fiir Versuchstierkunde (Hannover, Germany). In all experiments with
adult mice, 8-week-old animals were used. Newborns were bred in our department.
Organ preparation. For the preparation of the spinal cord, the spine was freed from adherent muscle tissue and
opened carefully, removing the dorsal part of the vertebrae until the spinal cord could be removed as a whole.
Other organs tested included brain, liver, spleen and blood. Dissected organs were disintegrated by ultrasonic
treatment and by freezing and thawing before titration of infectious virus.
lmmunosuppression, lmmunosuppression by cyclophosphamide was done as described by Rager-Zisman &
Allison (1976). A dose of 150 mg/kg of Endoxan (Asta-Werke AG, Bielefeld, Germany) was applied
simultaneously with the virus. Suppression of macrophage functions was achieved by the injection of 50 mg silica
suspended in isotonic NaC1 according to Zisman et al. (1970).
Mouse interferon. Mouse interferon was induced with Newcastle disease virus (NDV) in C-243 cells, by an
adaptation of the procedure developed by Cachard & DeMaeyer-Guignard (l 981). Briefly, monolayers of C-243
ceils in Petri dishes were incubated for 48 h in the presence of butyric acid (1 raM). After washing, monolayers were
infected with N DV (input m.o.i, of 2) and virus was allowed to adsorb for 2 h. The cells were then washed twice to
remove non-attached virus and incubated for 24 h in serum-free medium (MEM) supplemented with 3 mMtheophylline. Supernatants were harvested, centrifuged and stored at pH 2 for 5 to 7 days. This treatment proved
to be sufficient for total inactivation of NDV. Control preparations were performed identically, but without
adding NDV. The interferon preparation contained the three major protein species (35K, 28K and 22K), as
analysed by polyacrylamide gel electrophoresis. The antiviral activity of this interferon preparation was 5 x l0 s
IU/ml, hence having a specific activity of 1.6 x 107 IU/mg protein.
Test of natural killer (NK) cellactivity and interferon induction. For testing NK cell activity a 4 h s 1Cr release assay
was used with mycoplasma-free YAC-I lymphoma cells as targets (Engler et al., 1981). Interferon titrations were
performed exactly as described by Beck et al. (1980) using a one-step assay, L-cells and vesicular stomatitis virus.
RESULTS
R e s i s t a n c e o f various m o u s e strains to H S V - 1
ANG
We have shown previously that HSV-1 A N G does not cause lethal encephalitis in D B A / 2 and
C57BL/6 mice on intraperitoneal (i.p.) infection (Schrrder e t al., 1981). In the present study, we
have tested these and other inbred strains of mice including A / J , Balb/c and C3H, the
sensitivities of which to pathogenic HSV-1 strains, including HSV-1 W A L , have been shown
previously (Kirchner e t al., 1978). The results indicated that HSV-1 A N G was non-pathogenic
in all cases, even at doses as high as 2 × 107 p.f.u./mouse. It has been shown (Kirchner et al.,
1978) that D B A / 2 is highly susceptible to HSV-I W A L , and it was therefore chosen for most of
the experiments with HSV-1 A N G described here. We have confirmed that less than 10 2
infectious particles of HSV-1 W A L are required to cause lethal encephalitis after i.p. infection in
D B A / 2 mice.
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HS V strains differing in pathogenicity for mice
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100
60
5x 103
<
20
2
10~
5
I
6
8
4
Time post-infection (days)
5
1°"5
10
Fig. 1. Intracerebral infectionof DBA/2 mice with HSV-1ANG. Groups of at least 10 4- to 6-weeks-old
animals were individually infected i.c. with the indicated numbers of p.f.u. HSV-1 ANG suspended in
0-02 ml buffer.
Outcome of the infection by HSV-1 ANG when using different inoculation routes
For comparison, we tested the pathogenicity of HSV-1 A N G following (i) systemic routes, (ii)
peripheral routes and (iii) the intracerebral (i.c.) route of infection in DBA/2 mice. Whereas the
highly pathogenic strain HSV-1 WAL, used as a control, led to lethal encephalitis by any mode
of infection at 1 × 103 p.f.u./mouse, HSV-1 A N G proved to be non-lethal and did not cause
signs of illness when applied intraperitoneally, intravenously, intravaginally, orally, or via the
foot pads. However, i.c. infection with HSV-I A N G led to lethal encephalitis with very high
efficiency. The LDs0 for i.c. inoculation of HSV-1 A N G was close to 1 p.f.u. (Fig. 1). With some
individual HSV-1 A N G virus stocks, it appeared that even less than 1 p.f.u. (titre determined in
cell culture) might result in lethal infection after i.c. injection. This effect could be explained by
some HSV-I particles non-infectious in cell culture being able to replicate in mouse brain. The
physical to infectious particle ratios of individual HSV-1 pools used in this study normally
ranged between 10 : 1 and more than 100 : 1. (Schr6der & Urbaczka, 1978). Fig. 1 shows that the
time of death clearly depended on the virus dose.
As proof of the viral aetiology of the lethal outcome of i.c. inoculation of very low doses, virus
titres were determined in the central nervous system (CNS). Titres in the brains of mice that
eventually died after i.c. infection ranged between 5 × 103 and 1 × 104 p.f.u, per organ. In the
spinal cord, up to 105 p.f.u, per animal were found. Infectious virus was not demonstrable in the
CNS of survivors. Histological examination of spinal cord, spleen, liver, kidney, thymus, lung,
heart, intestine and testicles from DBA/2 mice after i.c. infection with HSV-1 A N G failed to
show major alterations in these organs. Focal haemorrhages around the corpus callosum, single
cell necrosis of the pyramidal cells, glia satellitosis and microfocal bleedings in the meninges
with inflammatory infiltrations suggested that the animals died from encephalitis or meningoencephalitis. All signs of myelitis were absent from tissue of the brain, the spinal cord and the
dorsal root ganglia. In control animals injected i.e. with saline, haemorrhages in the brain
indicated the site of injection, but other histological signs of the virus infection were absent.
Primary replication of HSV-1 ANG in the recipient mouse and the spread of infection
by the lethal and the non-lethal strains
The usual pattern of pathogenesis by HSV-1 A N G could be explained by assuming that this
virus strain is not able to replicate at the site of primary infection and therefore would not reach
the cells of the CNS. In tissue culture, we demonstrated that HSV-1 A N G could be propagated
in mouse primary fibroblasts and that it was able to replicate at the body temperature of the
mouse. To compare the spread of infection of HSV-1 A N G to that of HSV-1 WAL, virus titres
were determined in the organs of i.p.-injected DBA/2 mice. Fig. 2 shows the kinetics of virus
replication in the peritoneal exudate and the spleen. These two organs turned out to be the main
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G. K/JMEL
I
I
I
I
1
2
3
4
I
AND
(b)
OTHERS
I
I
I
I
I
I
i
3
4
5
6
7
e-~
_o
5 0
2
Time post-infection (days)
Fig. 2. Spread'of infection with (a) HSV-1 ANG and (b) HSV-1 WAL in DBA/2 mice. Groups of four to
six mice were infected intraperitoneallywith 4 × 105 p.f.u. At the times indicated, mice were dissected
and virus titres determined by independent titration of samples. The bars indicate the standard error of
the virus content per organ of peritoneal exudate (V), spleen (O), brain ([]) and spinal cord (11).
reservoir of virus in the mouse. In other organs (thymus. liver, kidney, lung, heart and lymph
nodes), virus was absent or ranged between 5 and 40 p.f.u, per organ in the first 6 days after
infection for both virus strains.
It was obvious that i.p. infection of DBA/2 mice with HSV-1 A N G is restricted, because after
the fifth day post-infection, virus could not be detected in any of the mouse organs. The i.p.
infection with HSV-1 W A L led to considerably higher titres (see Fig. 2b) in the peritoneum and
the spleen but not in the other organs tested. The course of infection paralleled that of the nonlethal strain as the virus was eliminated by the fifth day from peritoneum and spleen. Increasing
amounts of HSV-1 W A L were, however, found in C N S tissue 4 to 7 days after infection. The
difference in pathogenicity between the two virus strains is therefore not due to a general
restriction of primary replication.
W e tested whether a major difference in systemic transport of the virus would correlate with
the pathogenicity differences. After infection with either HSV-I strain, virus could not be
demonstrated by direct titration of blood or serum 1, 2, 3, 4 or 5 days after infection.
Thus, we are faced with the somewhat contradictory p h e n o m e n a that a HSV-1 strain
replicates to high titres in mouse organs after systemic or peripheral infection without
consequent lethal encephalitis, but that a few infectious virus particles invariably lead to lethal
encephalitis if administered intracerebrally. In the following parts of this section, studies of the
possible role of host defence mechanisms involved in the block o f the spread of HSV-1 A N G
infections to the C N S are described.
Infection of immunoincompetent mice with HSV-1 ANG
To investigate the role of age-dependent defence mechanisms, i m m u n o i n c o m p e t e n t newborn
mice were tested for susceptibility to HSV-1 A N G infection. For this study we used the C57BL/6
strain, because of its higher resistance to HSV (Zawatzky et al., 1982 a). C57BL/6 mice less than
7 days old were found to be susceptible to i.p. infection with HSV-1 A N G . Although the exact
value of the LDs0 was not established, less than 103 p.f.u, were found to be sufficient to kill all of
the animals (Table 1). The mechanism of the process limiting the spread of HSV-1 A N G into the
C N S after systemic or peripheral infection therefore depends on elements of the defence system
which are absent in newborn mice.
Homozygous nude mice (nu/nu) and, as control, heterozygous mice (nu/+) (in each case I0
animals per group) were tested for their susceptibility to i.p. infection with 4 x 106 p.f.u. HSV-1
A N G . Only about 1 0 ~ of the mice died in either group between 2 and 21 days after infection.
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I
I
1
2
I
I
I
I
1
3
4
5
6
7
319
5
4
,-"
4
3
1
Time post-infection (days)
Fig. 3. Spread of HSV-l ANG after immunosuppression with cyclophosphamide of DBA/2 mice.
Following injection of 150 mg/kg cyclophosphamide mice were injected with 4 x 10s p.f.u.
intraperitoneally. At the time points indicated, mice were dissected and virus titres determined by
independent titration of samples. Mean and standard error of virus titres per organ are given in the
figure for four to six mice per time point; parallel blood samples were pooled. ~', Peritoneal exudate;
O, spleen; O, liver; A, blood; m, brain.
Table 1. Mortality of newborn and adult C57BL/6 mice following i.p. infection with HSV-1 ANG
Expt. no. Age (days)
1
56
2
6
6
6
Dose used for infection
(p.f.u./animal)
Deaths/total number per group
2 x 106
0/10
2 × 104
6/6
2 x 103
6/6
2 x 102
2/6
Titration of the organs of those homozygous mice which died between these days failed to show
infectious virus in the brain or spinal cord. These results indicate that the resistance against
HSV-1 A N G is not dependent on mature T-lymphocytes.
Infection of adult mice with HSV-1 ANG following immunosuppression
F o r experimental immunosuppression D B A / 2 mice were treated with 150 mg/kg cyclophosphamide (Rager-Zisman & Allison, 1976). All of the immunosuppressed but uninfected animals
survived whereas after i.p. infection with 1 x 105 p.f.u. HSV-1 A N G , all of the animals died. To
examine the influence of immunosuppression on the course of infection, some of the infected
animals were killed at appropriate time points, dissected and examined for the presence o f
infectious virus in brain, spinal cord, liver, spleen and in the blood.
Figure 3 shows that cyclophosphamide exerted a prominent effect on the kinetics of the
infection. The drug induced a considerable increase in virus replication, particularly in the liver,
peritoneal cavity and spleen. Titration of blood samples showed a distinct viraemia. However,
little infectious virus was recovered from C N S tissue.
The restriction of infection with HSV-1 A N G at the site of p r i m a r y infection and the spleen
can clearly be overcome by immunosuppression. Unfortunately, cyclophosphamide changes the
pathogenesis so much that no conclusion can be drawn about differences between the two virus
strains, nor about a correlation between immunosuppression and the block in spreading o f HSV1 A N G to the CNS.
Injection of 2.5 g/kg silica was used to suppress macrophage activity in D B A / 2 mice. The
pretreated animals were infected i.p. 24 h later with 1 × 105 p.f.u. HSV-1 A N G . In this
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G. KfJMEL AND OTHERS
Table 2. Natural killer cell activity* of C57BL/6 mice after i.p. injection of H S V - I W A L or
HSV-1 A N G
Virus
Effector cell origin
~
r
strain
Spleen Peritoneal cavity
WAL
ANG
Uninfected
44
42
20
33
35
3
* Expressed as ~ specific lysis.
Table 3. In vitro sensitivity of HSV-1 strains A N G and W A L to interferon in the L-929 cell line,
or in mouse embryo fibroblasts (MEF) derived from DBA-2 or C57BL/6 mice*
L-929
F
Virus
HSV-I A N G
HSV-1 W A L
-
-
-
A
_
_
MEF-DBA
,
.
IFN
r
A
-
MEF-C57
~ ( . ~ . K
IFN
-
2-0 × 106 2.5 x 104 3.3 × l 0 s 4.5 x 104 4.0 x 105
3"1 × 100 6.5 x 104 4.5 x 105 7.5 × 104 4.0 × 105
•
IFN
2.0 x 104
2-8 x 104
* The values s h o w n are the progeny virus yield, g i v e n as the m e a n from three, 60 m m , Petri d i s h e s infected in
parallel at an m.o.i, of 2. T h e r e were 2-5 x 106 L-929 cells or 1-6 × 106 M E F cells p e r dish. C u l t u r e s were
p r e t r e a t e d w i t h 500 I U interferon ( I F N ) per m l m e d i u m .
experiment, only 2 out of 15 animals died. Thus, we conclude that the mechanism limiting the
spread of HSV-1 ANG into the CNS is not dependent on macrophage activity.
Analysis o f N K cell activity after injection o f H S V - 1 A N G
Major differences in the pathogenicity patterns of HSV-1 strains A N G and WAL could
possibly be due to a difference in the capacity to induce N K cells. The NK cell activity induced
after i.p. infection with HSV-1 A N G and the lethal strain HSV-1 WAL was assayed in C57BL/6
mice 24 h after i.p. infection with 1 x l0 s p.f.u, Table 2 shows that there were no differences in
NK cell activity regardless of whether spleen ceils or peritoneal exudate cells (PEC) were
examined.
Induction of and sensitivity to, interferon
The observed difference in the pathogenicities of HSV-1 A N G and HSV-I WAL could be a
consequence of either a difference in interferon induction or different sensitivities of the virus
strains to interferon. Primary embryonic mouse fibroblasts were cultured from the strains
DBA/2 and C57BL/6. Table 3 shows that strains A N G and WAL replicated with the same
efficiency in either of the two primary cell cultures or in the mouse cell line L-929 used as a
control. Pre-treatment of either cell culture with an interferon dose of 500 IU/ml culture medium
resulted in a reduced yield of progeny virus in both A N G and WAL. In each of the cell systems
used, the sensitivity of the two virus strains to interferon proved to be essentially the same.
In a mixed in vivo-in vitro approach, virus was injected i.p. and after one day PEC were
recovered and cultivated without further addition of virus for 24 h. Subsequently, interferon
titres in the tissue culture supernatant were determined. No difference between the virus strains
was observed (Table 4).
DISCUSSION
Resistance of mice to peripheral infection with HSV is controlled by host gene functions
(Lopez, 1975). Thus, it was important that our previous observation that HSV-1 A N G is nonpathogenic for C57BL/6 and DBA/2 mice (Schr6der et al., 1981) be extended to additional
inbred mouse strains, including some that are known to be exquisitely sensitive to i.p. infection
by other HSV strains (Kirchner et al., 1978).
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Table 4. Interferon production in peritoneal exudate cell cultures of C57BL]6 mice after i.p.
infection of pathogenic HSV-1 W A L and non-pathogenic HSV-1 A N G
Infecting virus
HSV-I WAL
HSV-I ANG
Mock-infected
Interferon titre
(IU/mlmedium)
450
420
10
One might expect that resistance of mice to infection with HSV-1 A N G is expressed at the
level of the target cells of lethal virus infection, i.e. the brain cells. We have shown, however, that
HSV-1 A N G causes a lethal encephalitis after i.c. infection. Furthermore, following i.p.
infection, a considerable amount of HSV-1 A N G can be found in the peritoneal cavity. Thus,
HSV-1 A N G obviously is not non-pathogenic in a strict sense. Virus titres of strain A N G in the
peritoneum and in the spleen are of a similar order of magnitude to those observed for HSV-1
WAL, indicating that the initial replication of HSV-1 A N G in vivo is not restricted. However, its
transport to the brain or its propagation in the central nervous tissue seem to be efficiently
blocked by a host defence mechanism.
Previous work in our laboratory on infection of mice with HSV-1 WAL has suggested that
interferon plays a critical role in the reistance of C57BL]6 mice to HSV (Zawatzky et al., 1981).
This conclusion is based on the correlation observed between high titres of early interferon at the
local infection site and high virus resistance (Zawatzky et al., 1982 b). DBA]2 mice did not show
this type of early interferon production. In the present study, however, we found that DBA/2
mice nevertheless resist even high doses of HSV-1 ANG. Series of in vitro and in vivo
experiments showed that HSV-1 A N G does not induce a more efficient interferon response than
HSV-1 WAL in DBA mice or that the non-lethal virus has a higher sensitivity to interferon.
A variety of additional approaches was utilized to study the role of defence mechanisms in the
lack of pathogenicity of HSV-1 ANG. First, it appeared that macrophages were not playing a
major role, since treatment of mice with high doses of silica did not remove resistance to HSV-1
ANG. Immunosuppression with cyclophosphamide led to a dramatic enhancement of the
invasiveness of HSV-1 A N G and to the death of the animals. It seems therefore safe to assume a
major role of immune control in the restriction of primary replication of HSV-1 at the site of the
inoculation and in the spleen. It cannot be decided from the data whether the second step of the
course of infection, transport to and propagation in CNS tissue, is under the control of the
immune system.
Newborn mice up to 10 days of age are also fully susceptible to infection with HSV-1 ANG.
Thus, the relevant defence mechanism that restricts the infection with HSV-1 A N G appears to
be absent during the first 10 days of life. Since we have previously shown that N K cell activation
by HSV is defective in newborn mice (Zawatzky et al., 1982a), we have measured activation by
HSV-1 A N G to test the assumption that HSV-I A N G is a better N K cell inducer than HSV-I
WAL. This was found not to be the case, however, so we assume that N K cells do not play a
major role in the restriction of HSV-I ANG.
We have demonstrated that nu]nu mice have a pronounced resistance to HSV-1 ANG.
Therefore we conclude tentatively that mature T-cells do not play a role in the resistance of mice
to infections with this strain of HSV. Nude mice are not only deficient in cell-mediated
immunity but also lack the capacity to produce certain antiviral antibodies (Burns et al., 1975).
Thus, nude mice are unable to produce IgG antibodies against HSV (Hilfenhaus et al., 1981).
Our data with HSV-1 A N G in nude mice therefore suggest that the IgG response of the host is
not responsible for the observed inhibition of pathogenicity.
In this context, it has to be remembered that primary virus replication is of the same order of
magnitude in the peritoneal cavity and in the spleen and that no viraemic process was found for
HSV-1 A N G or for the pathogenic strain HSV-I WAL. Thus it appears that the blocking
mechanism which prevents HSV-1 A N G from reaching the brain is not an early but, rather, a
relatively late defence mechanism. The studies of Kastrukoff et al. (1981) have indicated that
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G. KUMEL AND OTHERS
a n t i b o d i e s o f the I g M class are p r o d u c e d w i t h i n a few days o f i n f e c t i o n o f m i c e by H S V . Since
the p r o d u c t i o n o f I g M antibodies in n u d e m i c e is n o r m a l and does not require h e l p e r T-cells, one
possible e x p l a n a t i o n i n v o l v i n g the i m m u n e system would be t h a t HSV-1 A N G , in c o n t r a s t to
p a t h o g e n i c HSV-1 isolates, is a m o r e effective i n d u c e r o f I g M antibodies.
O n the o t h e r hand, unspecific, i.e. n o n - i m m u n e , processes h a v e to be c o n s i d e r e d as a possible
cause for the lack of p a t h o g e n i c i t y o f HSV-1 A N G . Differential r e p l i c a t i o n in special t a r g e t
cells, differential uptake by p e r i p h e r a l neurons or an unspecific, a g e - d e p e n d e n t process are
e x a m p l e s o f such n o n - i m m u n e m e c h a n i s m s . But diffferential r e p l i c a t i o n in m a c r o p h a g e s (J.
Briicher & H. K i r c h n e r , unpublished), a difference in v i r a e m i c transport, i n t e r f e r o n - s e n s i t i v i t y
or its induction, or N K cells all seem to be excluded.
Recently, an isogenic v a r i a n t o f HSV-1 A N G has been isolated in this l a b o r a t o r y w h i c h
p r o v e d to be highly p a t h o g e n i c for m i c e after p e r i p h e r a l i n f e c t i o n ( K a e r n e r et al., 1981). It
displays distinct alterations in its g e n o m e w h e n c o m p a r e d to the original HSV-1 A N G . A
c o m p a r a t i v e analysis of HSV-1 A N G and its p a t h o g e n i c v a r i a n t , w i t h r e g a r d to virus s p r e a d
and their influence on various host d e f e n c e m e c h a n i s m s , is in progress.
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