J. gen. Virol. (1986), 67, 387-390.
Printed in Great Britain
387
Key words: rabies virus/inter[eran]neutralization
Neutralization of Interferon Produced Early during Rabies Virus Infection
in Mice
By R. M A R C O V I S T Z , J. G A L A B R U , H. T S I A N G AND
A. G. H O V A N E S S I A N *
Ddpartement de Virologie, lnstitut Pasteur, 25 rue du Dr Roux, 75724 Paris COdex 15, France
(Accepted 4 November 1985)
SUMMARY
Inoculation of mice (strain C3H/He) with a purified preparation of fixed rabies virus
led to the production of interferon with two different peaks of activity detectable in the
plasma: an early peak 24 h after inoculation followed by another peak on the 7th day
after infection. The level of splenic 2-5A synthetase was enhanced in parallel with the
pattern of interferon activity. Neutralization of the first peak of interferon activity by
anti-mouse ct/fl interferon globulin blocked the induction of splenic 2-5A synthetase
and modified the development of disease. Infected mice given anti-interferon globulin
had a significantly shorter morbidity period than normally infected mice. These results
suggest that interferon produced early after virus inoculation plays a role in the onset of
rabies disease.
The production of interferon during rabies virus infection has been reported by several
investigators (Stewart & Sulkin, 1966; Wiktor et al., 1972; Marcovistz et al., 1984a). Inoculation
of mice with a mouse brain homogenate containing rabies virus (challenge virus strain : CVS)
results in the production of interferon with two different peaks of activity detectable in the
plasma: an early peak 24 h after inoculation followed by another peak during the terminal phase
of the infection. The specificity of the first peak of interferon activity has been controversial,
since it is also detectable in control mice injected with a brain homogenate from a normal
uninfected mouse (Marcovistz et al., 1984a). The second peak of interferon activity, however,
may be due to the presence of high levels of interferon produced in the brain in response to rabies
virus replication (Marcovistz et al., 1984a, b). Interferon produced during rabies virus infection
is stable at pH 2 and is neutralized by antibodies against mouse ~/fl interferon. This interferon is
active in infected mice as judged by the induction of pppA(2'p5'A)n synthetase (2-5A
synthetase) and poly(rI), poly(rC) Sepharose-bound protein kinase in different organs. Here,
we show that the first peak of interferon is also detectable in mice injected with a purified
preparation of fixed rabies virus from infected baby hamster kidney (BHK-21) cells. Rabies
virus is therefore capable of inducing interferon at the site of the virus inoculation, before its
migration to the central nervous system. This early peak of interferon production seems to play a
role in the development of infection, since mice inoculated with rabies virus and injected with
anti-mouse ~/fl interferon globulin showed a shorter morbidity period than the corresponding
control infected mice. Previously, antibodies against mouse ~/fl interferon have been used to
demonstrate the role of interferon in the pathogenesis of several acute virus diseases in mice
(Fauconnier, 1970; Gresser et al., 1976a, b, 1979, 1983; Rivi+re et al., 1977; Guillon & Tsiang,
1980; Saron et al., 1982; Pfau et al., 1983). However, this is the first report which provides
evidence to indicate that such antibodies actually neutralize interferon in the circulation. The
induction of 2-5A synthetase is inhibited in rabies virus-infected mice treated with anti-mouse
~/fl interferon globulin.
The CVS fixed strain of rabies virus, cultured on BHK-21 cells and purified by centrifugation
in a sucrose gradient (Arita & Atanasiu, 1980), was used in these experiments. Two-month-old
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388
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Fig. 1. The action of anti-mouse interferon globulin on the level of circulating interferon activity and
splenic 2-5A synthetase in mice inoculated with rabies virus. Male mice (C3H/He, Pasteur), 2 months
old, were inoculated with virus (CVS, 1 LDs0 into the footpad at time 0) and sacrificed at different times
as indicated. Sheep anti-mouse ~/fl interferon globulin (11 ; the antibody preparation added at 104-fold
dilution neutralized 5 units of interferon activity per ml) or sheep control immunoglobulin (O) was
injected intravenously (100 ~tl/mouse) at 1, 10 and 40 h after virus inoculation. (a) Kinetics of
appearance of interferon activity in the plasma of infected mice (mean of duplicate points from two
animals) injected with control immunoglobulin (O) or with anti-interferon globulin (11). (b) Kinetics of
induction of splenic 2-5A synthetase in infected mice injected with control immunoglobulin (O) or
with anti-interferon globulin (11). The 2-5A synthetase in spleen extracts was assayed after partial
purification on poly(I).poly(C)-Sepharose (Hovanessian & Rivi6re, 1980). The level of 2-5A
synthetase is expressed as nmol AMP incorporated into 2-5A per nag of spleen per h. The level of 2-5A
synthetase in the spleen of normal mice (uninfected) is shown at time 0.
1
6
mice were inoculated in the footpad. The infectivity of virus in brain extracts was titrated in
mice as described by Kaplan & Koprowski (1973). Antibody production against rabies virus was
titrated by the fluorescent focus reduction test (Sureau et al., 1982). Mouse interferon activity
was measured by inhibition of the cytopathic effect of vesicular stomatitis virus (VSV) on mouse
L-929 cells (Ito & Montagnier, 1977). The effect of interferon produced during rabies virus
infection was assessed by measuring the level of 2-5A synthetase in the spleen (Marcovistz et al.,
1984a). Anti-mouse ~/fl interferon globulin was obtained from a sheep immunized with mouse
a/fl interferon at a specific activity of 2.5 × 10s N I H units/mg protein (Galabru et al., 1985).
Inoculation of mice with a purified preparation of fixed rabies virus led to the production of
two peaks of interferon activity (Fig. 1 a). The early peak was detectable 24 h after virus
inoculation whereas the second peak reached its maximum value at the time of paralysis and
death of animals. In order to investigate the role of the early production of interferon in the
pathogenesis of rabies virus infection, we treated mice with anti-mouse a/fl interferon globulin
at 1, 10 and 40 h after virus inoculation. In such mice, the circulating interferon activity only
became detectable on the 4th day of infection and increased until death (Fig. 1 a). Thus, infected
mice which were injected with anti-interferon globulin, showed only one peak of interferon
activity which started 2 days earlier than the corresponding second peak of interferon in control
infected mice. This was probably because the development of rabies disease was accelerated by
neutralizing the first peak of interferon (see below).
In accord with the production of interferon, the level of splenic 2-5A synthetase was
enhanced in infected mice. Two peaks of enhancement were observed in control infected mice
on days 2 and 7 (Fig. l b). On the other hand, the level of 2-5A synthetase remained constant for
the first 3 days and thereafter increased in infected mice injected with the antibodies (Fig. 1 b).
These results indicate that interferon produced during rabies virus infection is active at least for
the induction of 2-5A synthetase. The lack of enhancement of 2-5A synthetase (during the first
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389
Table 1. Effect of neutralization of the first peak of interferon activity on the development of rabies
virus infection in mice*
Treatment
None
Control IgG
Anti-IFN IgG
Day of onset
of paralysis#
5.0 _+ 0
5.2 + 0.4
(P, > 0-5)11
4.6 + 1.1
(ez > 0"3)I1
Day of death#
7.4 +_ 0-5
7.6 _+ 0.5
(e~ > 0.5)
5.8 + 1-3
(Pz < 0.02)
Antibody
activity (IU)$
13
13
Virus infectivity
(log10 LDs0/ml)§
6.0, 6.2, 7.6
6.8, 7.0, 7.5
13
5.8, 7.5, 6.3
* Three groups of five male mice were infected with rabies virus as described in the legend to Fig. 1. The control
IgG and anti-IFN (interferon) IgG groups received three injections intravenously (1, 10 and 40 h) of sheep
control immunoglobulin and sheep anti-mouse c~/flinterferon globulin, respectively (as in the legend to Fig. 1).
Mice were examined daily and separated as soon as the first signs of paralysis became apparent. Blood and brain
were collected from mice with general prostration.
t Mean + standard deviation.
:~Average activity from three mice.
§ Virus infectivity from three mice.
IIel, Difference between mice injected with sheep control immunoglobulin and untreated mice; P2, difference
between mice injected with sheep anti-mouse ct/fl interferon globulin and untreated mice,
3 days) in mice injected with anti-interferon globulin demonstrates the neutralization of
interferon. In these experiments, the quantity of anti-interferon globulin given to infected mice
was just sufficient to neutralize the first peak of interferon. In an attempt to neutralize the
second peak of interferon, infected mice were treated with the antibody on days 3, 4 and 5 after
virus inoculation. However, in these latter experiments, only 2 0 ~ of the circulating interferon
activity was neutralized (data not shown). It should be noted that very high levels of interferon
are produced during the replication of rabies virus in the brain (Marcovistz et al., 1984a).
The pathogenesis of rabies virus infection in mice inoculated with the CVS strain (1 LD5o) is
characterized by paralysis of the posterior limbs on day 5. This infection is lethal for all mice by
day 7 or 8. Virus becomes detectable in the brain by immunofluorescence on day 4. Interferon of
type e/fl and antibodies specific against rabies virus are produced during the disease (Fig. 1 and
Table 1). Neutralization of the first peak of interferon activity accelerated the development of
the disease. Infected mice which received anti-interferon globulin had a morbidity period which
was significantly (P < 0.02) less than that for the control infected mice. On the other hand, the
activity of antibody against rabies virus in the plasma and infectivity of virus in the brain were
similar for both groups (Table 1). These results indicate that interferon produced early during
the course of the disease may play a role in the onset of the infection. This effect probably occurs
soon after virus inoculation and during the extraneural phase of the disease. The replication of
virus started on day 4 after inoculation when the first peak of interferon activity had passed.
When infected mice were treated with anti-interferon globulin, then high virus infectivities were
found on days 4 and 5. In these mice, therefore, virus replication started at least by day 3. It
should also be noted that enhancement of plasma interferon activity during the period of virus
replication in the brain was different in the two experimental groups (Fig. 1 a). Interferon
activity was detectable 2 days earlier in mice treated with anti-interferon globulin.
All these experiments were done with C3H/He mice. Among other strains of mice tested,
A K R mice showed a higher sensitivity to rabies virus infection than strains C3H/He, DBA/2 or
BALB/c. Interestingly, only A K R mice failed to produce the first peak of interferon activity.
The kinetics of the production of interferon activity and the development of the infection were
similar in C57BL/6, DBA/2, BALB/c and C3H/He mice.
Rabies virus-infected mice die in the presence of very high levels of interferon activity in the
circulation (the second peak) and the brain (Marcovistz et al., 1984a). This interferon seems to
be ineffective in inhibiting replication of virus since infected mice do not survive. It might be
that interferon is produced in insufficient amounts or is produced too late in the infection to
exert an efficient antiviral effect in the brain. Furthermore, it is possible that the protective
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390
Short communication
a c t i o n o f i n t e r f e r o n is i n h i b i t e d d u r i n g r a b i e s virus infection. It should be e m p h a s i z e d also t h a t
t h e d e v e l o p m e n t o f r a b i e s d i s e a s e d e p e n d s o n m a n y i n t e r a c t i n g f a c t o r s s u c h as t h e host i m m u n e
r e s p o n s e (Guillon & Tsiang, 1980; S m i t h et al., 1982). Thus, t h e first p e a k o f i n t e r f e r o n activity
m a y control the o n s e t o f rabies i n f e c t i o n w h e r e a s the effect o f t h e s e c o n d p e a k o f i n t e r f e r o n
activity m i g h t be m a s k e d by the i m m u n o p a t h o l o g i c a l r e s p o n s e o f i n f e c t e d mice.
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