J.
gen. Virol. (1983), 64, 1827-1830. Printed in Great Britain
1827
Key words: interferon~lymphocytic choriomening#is virus~interferon antibody"
Lethal Role of Interferon in Lymphocytic Choriomeningitis Virus-induced
Encephalitis
By C H A R L E S J. P F A U , 1. I O N G R E S S E R 2 AND K A T H E R I N E D. H U N T 1
1Department o f Biology, Rensselaer Polytechnic Institute, Troy, New York 12181, U.S.A. and
2Laboratory o f Viral Oncology, Institut de Recherches Scientifiques sur le Cancer, PBS, 94802
Villejuif France
(Accepted 29 March 1983)
SUMMARY
After intracerebral inoculation of adult C3H mice, the 'docile' strain of lymphocytic
choriomeningitis (LCM) virus multiplied to high titre in several visceral organs.
Although the virus content of lung, liver, spleen and brain was high, these mice did not
die but became long-term carriers of the virus. Injection of mice with the same dose of
the 'aggressive' strain of LCM virus resulted in much lower virus titres in these organs;
nevertheless, 100% of the mice died within 7 to 9 days. The results presented here show
that mice infected with the 'aggressive' virus do not die if treated with anti-interferon
globulin. Under these conditions the titres of 'aggressive' virus were as high in the
different organs as in mice injected with the 'docile' virus. These results are consistent
with the hypothesis that inhibition of LCM virus multiplication in various organs by
interferon results in a lethal disease. The possible mechanisms underlying this
seemingly paradoxical phenomenon are discussed.
It is generally held that interferons play an important role in recovery from acute primary
virus infections. One way of demonstrating this is by use of antibody to interferon.
Neutralization of virus-induced interferon results in rapid and extensive virus multiplication
and early onset of disease in a number of murine virus infections (Gresser et al., 1976a, b).
However, the studies presented here using anti-interferon globulin show that in lymphocytic
choriomeningitis (LCM) virus infection of adult mice, interferon does not protect the mice but is
somehow responsible for the lethal outcome of virus infection.
Two strains of LCM virus were used in these studies. Adult mice infected intracerebrally with
the 'aggressive' strain usually died with a convulsive central nervous system (CNS) disease 7 to 9
days after infection. Most mice infected intracerebrally with the 'docile' strain did not die
(Jacobson & Pfau, 1980) but developed a life-long ( > 9 months; C. J. Pfau, unpublished
observations) persistent infection. Paradoxically, the 'docile' virus multiplied to much higher
titres in the brain and in the visceral organs than the 'aggressive' virus (Pfau et at., 1982b).
Interferon seemed to play a pivotal role in influencing both virus multiplication and the
evolution of the disease. Thus, mice injected with the 'docile' virus developed CNS disease when
injected at appropriate times with inducers of endogenous interferon (Jacobson et al., 1981),
while mice infected with an 'aggressive' strain were spared by injection with anti-interferon
globulin (Saron et al., 1982a). In the latter experiments the titres of virus in the blood were
increased 100- to 1000-fold. The following experiment was designed to determine the role of
endogenous interferon in the evolution of disease induced by the 'aggressive' strain, and
specifically to examine whether administration of anti-interferon globulin to mice injected with
the 'aggressive' strain would result in a pattern of disease observed in mice injected with the
'docile' strain.
C3H mice were injected intracerebrally with 300 p.f.u, of either the 'aggressive' or 'docile'
LCM virus strains (Pfau et al., 1982 a). Thirty-four mice were infected with the 'aggressive' virus
and divided into three groups. The first group (10 mice) served as the untreated controls, while
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1828
Short communication
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Fig. 1. LCM virus content in tissues of mice injected with anti-interferon serum. After blood was taken
from the ophthalmic venous plexus the lungs (a), liver (b), spleen (c), brain (d) and kidneys (e) were
aseptically removed daily from one infected mouse in each of the following groups: aggressive virus +
anti-interferon globulin (O); aggressive virus + normal sheep globulin ([7); aggressive virus alone
(O); docile virus alone (I). Pooled sera (f) were treated as described in the text; the organs were
washed, weighed, ground with sterile sand, and resuspended in tissue culture media containing 10~
foetal bovine serum. Samples were centrifuged at low-speed and the supernatants stored at - 70 °C
until titrated by plaque assay (Jacobson et al., 1979).
the second and third groups (12 mice each) received either anti-interferon globulin or normal
sheep globulin. A fourth group (10 mice) served as the 'docile' virus controls. For the first 5 days
after infection, blood, brain and visceral organs were removed daily from one mouse in each of
the four groups. On the third day after infection and 2.5 h after injection of either anti-interferon
globulin or normal sheep globulin, two mice were exsanguinated from each of these two groups.
The sera within each group were pooled, dialysed to p H 2, redialysed to p H 7 and assayed for
interferon (Jacobson et al., 1981). Interferon was not detected ( < 16 International Units, IU) in
mice infected with 'aggressive" virus and treated with sheep anti-interferon globulin. Fig. 1
shows that the titres of 'aggressive' virus in antibody-treated mice were comparable to those in
untreated mice injected with 'docile' virus. Under these circumstances the ordinarily lethal
'aggressive' virus infection was benign, again comparable to the outcome of infection of mice
with the 'docile' virus (Table 1). On the other hand, the serum interferon titre was high
(1024 IU/inl) in mice infected with 'aggressive' virus and treated with normal sheep globulin.
These mice died and the virus titres in most visceral organs were low (Table 1, Fig. l).
When the experiment was terminated at 42 days post-infection, all mice had virus in their
blood. The five mice infected with 'aggressive' virus and treated with antibody (Table 1) had
titres of virus ranging from 0.6 x 103 t o 2"8 × 103 p.f.u./ml. The four mice infected with
'docile' virus (Table 1) had titres ranging from 2.3 × 105 to 9.4 x 105 p.f.u./ml. As there is no
great difference in the growth rates of these two virus strains in tissue culture (Jacobson, 1980)
the explanation for the 300-fold difference in titres in vivo may be that the aggressive virus
induces a continuous production of interferon (Saron et al., 1982b).
We had previously observed that intracerebral inoculation of very high concentrations of
'aggressive' virus mimicked the behaviour of 'docile' virus, and did not induce fatal C N S
disease. On the other hand, intracerebral inoculation of very low concentrations of'docile' virus
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Short communication
1829
Table 1., Effect of anti-interferon globulin on the evolution of disease in mice infected with the
'aggressive' ( A) and 'docile' ( D) strains of L C M virus*
Virus
strain
Treatment
A Untreated control
A Normal globulin
A Anti-interferon
globulin
D Untreated control
No. of dead mice/
no. usedt
5/5
5/5
Mean no. of days till
death (range):~
8.4(8-9)
8.7(8-9)
0/5
t/5
19.0
* Three-week-old female C3HeB/FeJ mice (Jackson Laboratories, Bar Harbor, Me., U.S.A.) were injected with
virus and observed daily. Beginning on the fifth day after infection the mice were spun by the tail to hasten
convulsive death. Mice received interferon antibody 0.5 h before, as well as 72 h after infection with aggressive
virus. Each intraperitoneal injection consisted of 0.1 ml of a 1 : 3 dilution of globulin taken from sheep immunized
with mouse interferon (Gresser et al., 1976a). This globulin had been extensively absorbed on mouse C-243 cells as
well as on a pool of mouse thymocytes and splenocytes. Its neutralizing titre was 1:400000 against 8 IU of C-243
cell mouse interferon. The same treatment schedule was followed for the group receiving normal (absorbed) sheep
globulin.
t Number of dead mice does not include deliberately sacrificed animals.
:~The experiment was terminated 42 days after infection.
resulted in low virus titres and was lethal (Pfau et al., 1982b). Thus, infection with the 'docile'
virus can under some circumstances result in death. There a p p e a r s to be, therefore, an inverse
relationship between the rapidity and the extent of virus multiplication in the b r a i n and visceral
organs and death. As death in L C M virus disease is attributed to the d e v e l o p m e n t of virusspecific thymus-derived cytotoxic lymphocytes (Cole et al., 1972), we m a y formulate the
following question. W h a t is the relationship between high virus titres (induced by 'docile' virus
or 'aggressive' virus in mice treated with anti-interferon globulin) and the failure o f virusspecific T lymphocytes to induce a lethal disease? W e know that the 'docile' virus induces a
virus-specific T lymphocyte response as well as the 'aggressive' virus (Pfau et al., 1982a).
Moreover, when mice were given fully functional virus-specific T cells by adoptive transfer
shortly after infection with 'docile' virus at a time when their own T cell response to the infection
had not reached detectable levels, they died with convulsions 5 to 6 days later (Pfau et al.,
1982b). We propose that high titres of virus in multiple target organs m a y cause dispersion of the
pool of cytotoxic T lymphocytes, thus preventing a focused (lethal) T lymphocyte concentration
in the brain.
Whatever the mechanism, our results show that when endogenous interferon induced by
'aggressive' virus is neutralized by anti-interferon globulin, the virus multiplies to high titres as
in mice infected with the 'docile' virus, and the mice now survive infection. Interferon thus
appears to be associated in some m a n n e r with lethality in L C M virus-infected adult mice. It has
previously been shown that endogenous interferon induced by L C M virus was also directly
responsible for a lethal syndrome in suckling mice (Riviere et al., 1977) a n d the d e v e l o p m e n t o f
glomerulonephritis in adult mice surviving neonatal infection (Gresser et al., 1978). The
relevance of all these studies on L C M virus infection in mice to h u m a n disease has yet to be
determined, but it is possible that under some circumstances in m a n interferon m a y exacerbate
rather than alleviate disease.
This work was supported in part by grants from the U.S. National Science Foundation (PCM 7917935); the
National Institutes of Health (U.S. Public Health Service grant AI-16562); D.R.E.T. (contract 80-34-522);
I.N.S.E.R.M. (CRL 81 20 08) (PRC 12 70 12); C.N.R.S. (ATP Etats-Unis); and the Richard Lounsbery
Foundation.
REFERENCES
COLa, 6. A., NAaa-L~NSON,N. & PRm,TDERGAST,R. A. (1972). Requirement for 0 bearing cells in lymphocytic
choriomeningitis virus-induced central nervous system disease. Nature, London 238, 335-337.
GRESSER, I., TOVEY, M. G., BANDU, M.-T., MAURY, C. & BROUTY-BOYE, D. (1976a). Role of interferon in the pathogenesis of virus diseases in mice as demonstrated by the use of anti-interferon serum. I. Rapid evolution of
encephalomyocarditis virus infection. Journal of Experimental Medicine 144, 1305-1315.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 01:48:26
1830
Short communication
GRESSER,I., TOVEY,M. G., MAURY,C. & BANDU,M.oT. (1976b). Role of interferon in pathogenesis of viral diseases in
mice as demonstrated by the use o f anti-interferon serum. II. Studies with herpes simplex, Moloney sarcoma,
vesicular stomatitis, Newcastle disease, and influenza viruses. Journal of Experimental Medicine 144, 13161323.
GRESSER, I., MOREL-MAROGER,L., VERROUST, P., RIVIERE, V. & GUILLON, J.-C. (1978). Anti-interferon globulin
inhibits the development of glomerulonephritis in mice infected at birth with lymphocytic choriomeningitis
virus. Proceedings of the National Academy of Sciences, U.S.A. 75, 3413-3416.
JACOBSON,S. (1980). Propertiesof in vitro and in vivo long term persistent infections with lymphocytic choriomeningitis
virus. Ph.D thesis, Rensselaer Polytechnic Institute, Troy, New York.
JACOBSON, S. & PFAU, C. J. (1980). Viral pathogenesis and resistance to defective interfering particles. Nature,
London 283, 311-313.
JACOBSON,S., DtnXO, F. J. & I'FAU, C. J. (1979). Determinants of spontaneous recovery and persistence in M D C K
cells infected with lymphocytic choriomeningitis virus. Journal of General Virology 44, 113-121.
JACOB,SON,S., FRIEDMAN,R. M. & PFAU, C. J. (1981). Interferon induction by lymphocytic choriomeningitis viruses
correlates with maximum virulence. Journal of General Virology 57, 275-283.
PFAU,C. J., VALENTI,J. K., JACOBSON,S. & PEVEAR,D. C. (1982a). Cytotoxic T cells are induced in mice infected with
lymphocytic choriomeningitis virus strains of markedly different pathogenicities. Infection and Immunity 36,
598-602.
PFAU, C. J., VALENTI,J. K., PEVEAR,D. C. & HUNT, K. D. (1982b). Lymphocytic choriomeningitis virus killer T cells
are lethal only in weakly disseminated infections. Journal of Experimental Medicine 156, 79-89.
RIVIERE, Y., GRESSER,L, GUILLON, J.-C. & TOVEY, M. G. (1977). Inhibition by anti-interferon serum of lymphocytic
choriomeningitis disease in suckling mice. Proceedings of the National Academy of Sciences, U.S.A. 74, 21352139.
SAP,ON, M.-F., RIVIERE, Y., GRESSER,L & GUILLON, I.-(2. (1982a).Prevention par les globulines anti-interferon de la
mortalite des souris infectees par le virus de la choriomeningite lymphocytaire. I. Lesions histologiques, titres
seriques du virus et de l'interferon. Annales de Virologie 132 E, 241-253.
SARON,M.-F., RIVlERE,Y., HOVANESSIAN,A. G. & GUILLON, J.-C. (1982b). Chronic production of interferon in carrier
mice congenitally infected with lymphocytic choriomeningitis virus. Virology 117, 253-256.
(Received 10 January 1983)
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