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Stress-induced neuroinvasiveness of a neurovirulent noninvasive Sindbis virus in cold or isolation subjected mice
Life Sciences, Vol. 48, pp. 1493-1500 Printed in the U.S.A.
"
Pergamon Press
STRESS-INDUCED NEUROINVASIVENESS OF A NEUROVIRULENT NONINVASIVE SINDBIS VIRUS IN COLD OR ISOLATION SUBJECTED MICE D. B e n - N a t h a n Dept.
S. L u s t i g
of Virology, Israel Institute for P.O.Box 19, 70450 Ness-Ziona, (Received
in final
form February
H.D.
Danenberg
Biological Israel 8,
Research
1991)
Suw~ar 7 Effects of cold or isolation stress on brain penetration by the neurovirulent noninvasive Sindbis virus strain (SVN) w e r e s t u d i e d i n m i c e . SVN i n j e c t e d intracerebrally (i.c.) causes acute encephalitis and kills adult mice but is unable to invade the brain and kill when injected intraperitoneally (i.p.). Mice inoculated i . p . w i t h SVN w e r e e x p o s e d t o c o l d s t r e s s o r were singly housed. Both stress patterns induced SVN encephalitis and death in 42% (cold) and 37% (isolation) of the tested mice. No death was observed in the control injected miceg Brain virus levels were found to be more than i0- PFU in all dying mice. No virus was detected in the control group brains. The virus that was isolated from the brains of moribund mice demonstrated no changes in neuroinvasive and neurovirulent properties. We suggest a stress induced blood-brain-barrier opening with subsequent virus entrance as the mechanism of stress induced SVN encephalitis. Stress is a term defining the reaction of the body to a variety of emotional and physical stimuli which endanger homeostasis. Studies have demonstrated the intimate connections between stress and the immune system (1,2,3). The stress effects on the immune system in human and animal studies are most commonly suppressive, with changing production and activity of a wide range of immune system components. Viruses are known to flourish in the stressed host, which suffers increased morbidity and mortality (4,5,6,7,8). Studies concerning neurovirulent viruses report of increased morbidity and mortality as a consequence of stress treatment (9,10). As a rule, it is believed that at times of immunosuppression there is an increased rate of viral infection of the central nervous system. This phenomenon is explained either by persistance and reactivation of the virus in the brain or increased multiplication in the periphery of a virus with neuroinvasive properties (ii). Another mechanism
which
can
be
responsible
for
the
0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
increased
rate
of
1494
Stress-Induced Vira~ Brain Invasion
Vol. 48, No. 15, 1991
encephalitis is the disruption of the blood-brain-barrier and subsequent virus penetration. Blood-brain-barrier permeability to [131]-Iodlne-sodium was found increased at times of stress (12). Histological study of immobilized rat brains revealed microvascular damage in the midbrain (13). West Nile Virus-25 (WN-25) is an attenuated nonneuroinvaslve strain of West Nile Virus that does not produce any detectable viremla. Previous work studying the effect of stress on mice inoculated with WN-25, demonstrated changes in viral traits, resulting in the recovery from the brain of virus with neuroinvasive properties comparable to the neurolnvasive wild type WNV (14). Those remarkable results did neither reject or support the theory of stressinduced encephalitis due to blood-brain-barrier breach. The present study was planned to check the influence of stress on brain invasion by a noninvasive neurovirulent virus, that produces a high viremia titer and is not derived of a neuroinvasive virus. Sindbis virus neuroadapted strain (SVN) is an alphavirus not invading the brain in mice when injected intraperitoneally (i.p.), but causing encephalitis when injected intracerebrally (i.c.), even when in a very low dose (15,16 and S. LUSTIG, M. HALEVY, D. BEN-NATHAN, Y. AKOV - submitted for publication). Mice inoculated i.p. with SVN were subjected to various kinds of stress and checked for changes in mortality and virus levels in blood and brain tissue as well as other stress parameters. Materials and Methods Sindbls virus - neuroadapted strain (SVN) A Sindbis virus (SV) was isolated in 1983 from a pool of culicine mosquitoes gathered in southern Israel. It was lethal to suckling mice by all routes of injection, but without ill effects to weanling mice (outbred, ICR). This SV strain was passed consecutively by i.c. injection in suckling mice. A new passage variant (SVN) was isolated, which caused death in weanling mice in~ected i.c., but not in those injected i.p. Stock v~rus contained 7.5xi0 plaque forming units (PFU\ml). The i.c. titer was 3.4xi0" MICLD50kml. The stock virus was kept at -70°C. Mice Charles river mice CrI:CDR-I (ICR) female were obtained at the age of 21 days (10-12 g body weight) and kept in our vivarium until the age of 27 to 42 days. In all studies, mice of the same age and batch were used. Mice were divided into groups following inoculation and were observed for mortality until the end of the experiment (21 days). For virus level in the brain and for lymphoid organs weight, mice were sacrificed 7 days after inoculation. Virus inoculation Each mouse was inoculated i.p. with 1.5x105 PFU of SVN. The virus dilutions were performed using inactivated rabbit serum (10%) in 0.9% saline containing penicillin(l,000~/ml). Groups of 6-10 mice were used, and the results were calculated according to the method of Reed and Muench (17). Stress Cold stress Mice were placed for 5 minutes in 3 cm deep ice cold water (I±0.5°C). Mice were exposed to cold stress either twice, on inoculation day and the day after, or five times until 4 days post inoculation. Isolatlon Mice were housed in individual cages immediately after inoculation until the end of the experiment. Control mlce were housed 6 per cage. Veto cells The Veto cell line was derived from kidneys of normal African Green Monkeys. The cells were grown in Dulbeco's Modified Eagle Medium (DMEM) containing 10% fetal calf serum (FCS).
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Stress-Induced Viral Brain Invasion
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Isolation of SVN from the brain of infected mice Each brain was rinsed in cold PBS and sonlcated, The virus suspension was centrifuged at 3,000 rpm for I0 min. The supernatant was alllquoted into plastic tubes and stored at -70°C until further processes. The virus level in the brain was determined by titration of virus in Veto cell line. Isolation of SVN from blood Cold stressed and control mice were bled at various time points from the tail vein into special test tubes (brand serum separator, Beckton Dickinson). Virus content in the serum was plaque-assayed in Veto cells. Organs weight Mice were individually weighed 7 days after inoculation. The mice were sacrificed and the thymus and spleen were removed immediately and weighed aseptically. Titration of virus in tissue cultures For demonstration of SVN plaques in Veto cells the original plaque technique of Dulbeco and Vogt (18) was used. A dilution of virus was added to the Vero cell monolayers in petri dishes and incubated at 37°C for lh to permit viral adsorption. The monolayer is overlaid with MEM x 2 and tragacanth (Gum tragacanth Grade III G-I128, Sigma) containing 2~ FCS and 2.4~ NaHC0 R. The cultures were incubated (37°C,5~C02) for 72h. Plaques were counted ~fter staining the monolayer with neutral red (0.05~). The same procedure was followed for BHK cells. All plaques were counted by an experienced investigator. In vlvo assay The virus isolated from the brain of dying mice was grown in Veto cells and injected i.p. or i.c. to non stressed mice. Mice were observed for mortality for a period of 21 days. Data analysis Results are expressed as means±SEM. The data were analyzed by one way ANOVA or Fisher exact probability test. Results The effect of stress on body temperature. Body temperature of mice exposed to cold water declined sharply to a temperature of 14°C and returned to a normal level about an hour and a half later. Results are shown in figure i. Isolation stress had no effect on body temperature compared to the control group. The effect of cold or isolation on body and lymphatic organ weight. To study the general effect of stress on mice, spleen and thymus weights were measured 7 days after SVN inoculation and exposure to stress. Isolation and cold stress both reduced spleen and thymus weight. SVN inoculation of non stressed mice induced a non significant reduction of lymphoid organs weight. Inoculation of stressed mice with SVN induced a significant weight reduction. The results are presented in figure 2. The e f f e c t of cold or isolation stress patterns on mortality of mice inoculated with SVN. These experiments were done to determine the effect of cold or isolation stress on mortality of mice after inoculation wi~h SVN. All groups except the control i.c. group were inoculated i.p. with 2x10- PFU of SVN. Results are presented in table I. The virus was proved to kill non-stressed mice only by the i.c. route, no mortality was observed in the control group that was injected i.p.. Cold stress, either two or five exposures, induced a mortality rate of 20% (2 exposures) to 42~ (5 exposures). The mortality rate of the single housed mice was 37~ (p<0.05 in all groups compared to control group). Death in all stress groups occurred on days 7,8,9 following inoculation with no significant difference in the mean time of death.
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Stress-Induced Viral Brain Invasion
40
Vol. 48, No. 15, 1991
~
7
35 30 m
o
E=
D
4 , 3
15 2
10 oz
÷
5
25 20
t--
spllen/B.W.
~h~ thymus/B.W.
1
5 II,],IIIIL,I,I,I,
II
i,i,i,i,i
;3 -'0 30 40 50 60 70 80 9 0 1 0 0 1 1 0 ~20
240
conlrol
SVN
Time after exposure to cold water (rain)
cold
treatment
cold
+ SVN
Isolation
Isolation
÷SVN
group
FIGURE 2
FIGURE 1
Body temperature of mice exposed to cold water (n=9)
Ratio of lymphoid organ to body weight (xlO00) 2<0.05 TABLE
1
The effect of cold or isolation stress on mortality of SVN inoculated mice.
Treatment group
a
Mortality
D/T Control i.p. Control i.c 5 Cold 2 days b Cold 5 days Isolation
0/24 24/24 4/20 11/26 14/38
% 0 i00 20~ 42~ 37-
a
All groups, except the i.c. group, were injected i.p. with 2x105 PFU of SVN, control i.c. group were injected i0 PFU (0.03ml) of SVN. b - 5min at I±0.5°C immediately after inoculation, and on the day after (2d), and on the 4 consecutive days (5d). c - R<0.05 d - 2<0.01 D/T - Dead/Total. Virus level in the blood of stressed and non stressed mice Figure 3 illustrates virus level in the blood of cold-stressed and in nonstressed mice. Virus levels reached a peak at 24h post inoculation and than declined with no virus in the blood at 72h post inoculaion. The virus level in the stress group was less than a log higher than the level in the control group. Virus level on brain 7 days after inoculation and exposure to stress To determine brain virus level in stressed infected mice, brains were taken from moribund mice on day 7 after inoculation. As a control, brains were taken from non stressed infected mice. The results are presented in table 2. No virus was detected in brains of non-stressed injected mice, while in brains of moribund mice a significant virus titer was found.
Vol. 48, No. 15, 1991
Stress-Induced Viral Brain Invasion
1497
Inoculation of mice with the virus recovered from brains of isolation or cold stressed m i c e . In order5to study changes in neurolnvaslveness, 24 mlce were inoculated i.p. with 5xlO of virus isolated from brains of stressed mice (12 - isolation and 12 - cold ). 21 days of observation for mortality recorded no death. Brains taken from 6 mice at day 7 following inoculation were free of virus. Intracerebral inoculation of mice with the isolated virus had similar results to the original SVN virus.
Cold
•
-
SVN
+
0
-
SVN
Control
;
,
T
1
2
3
-
?
Days post inoculation
FIGURE 3 Viremia level in cold stressed mice (n=6)
TABLE 3
TABLE 2 The e f f e c t o f c o l d o r I s o l a t l o n on b r a i n SVN l e v e l a t 7 d a y s a f t e r i n o c u l a t i o n and e x p o s u r e t o s t r e s s .
The effect of i.c. PBS injection on mortality of mice inoculated peripherally w l t h SVN.
Treatment group
N
log10 PFU/brain
Treatment group
Control i.p. Control i°c. Cold (5 days) Isolation
8 6 8 8
~2 6.89±0.45 6.51±0.31 6.73±0.27
All groups except the isC. group were injected l.p. with 2xlO PFU of SVN. i.c. group were injected with i0 PFU. p
Mortality D/T
PBS SVN SVN SVN
i.c. only~ i.p.(5xlO-pfu) i.c.(lO pfu) i.p.+PBS l.c.
018 0116 818 14/16
0 0 I00 87.5
0.03ml of PBS injected i.c. 24h after inoculation.
I n j e c t i o n o f s a l i n e s o l u t i o n t o t h e b r a i n o f m i c e i n o c u l a t e d w i t h SVN. Table 3 presents the effect of mild mechanical trauma on the penetration of noninvaslve virus to the brain. Intracerebral injectlon'of O.03ml of PBS to mice 24h post inoculation with SVN (time of peak viremla) induced encephalitis and mortality in 87.5% of the mice. Injection of PBS to noninfected mice did not cause any mortality.
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Stress-Induced Viral Brain Invasion
Vol. 48, No. 15, 1991
Discussion The present study demonstrates an encephalitis inducing effect of stress on mice inoculated with a neurovirulent virus with no neuroinvasive properties. The stress patterns were designed to simulate two major kinds of stress, psychological and physical. While the psychological i.e. isolation stress, was of prolonged duration, the physical cold stress was of short term, applied either twice or several times. The results clearly exhibit similarity in stress effect on lymphoid organ weight, a common parameter to the general immunosuppressive properties of stress. The effect is attributed to elevated corticosterone levels and is known to be augmented in viral infection (19). As well as for lymphoid organ weight, the results clearly exhibit similarity in SVN encephalitis rate and mortality in the different stress exposed groups. Thus, suggesting a common mechanism of influence activated by various stress patterns which differ in type and duration. Single housing effects on the immune system are controversial, both increased (20,21) and decreased (2,22) susceptibility to disease were observed in mouse and rat models. The changes in the effect are most probably time-dependent with an immunosuppressive effect changing with time to immunoenhancement. Our previous experience with short-term isolation stress in mice is clearly in favor of an increased infection due to an immunosuppressive effect (i0,14). There are three known pathways of virus spread to the brain - hematogenous, neural, and via the olfactory nerve (23). The hematogenous spread is the one commonly taken by alphaviruses. The mechanism by which stress induces Sindbis virus neuroinvasiveness is not clear. However, it can be explained by three major different hypotheses. Suppressed immmune response in stress, with defective function of macrophages and lymphocytes, leads to enhancement of viral replication in the blood and peripheral organs, induction of a selection process, and selection of a neuroinvasive strain. This hypothesis seems valid in the case of the neuroinvaslveness change of the attenuated WNV strain (14). In the present study the virus recovered from brains of stress-subjected mice did not show any changes in the neuroinvasive properties, thus 8nnuling this theory. The second theory is also based on the enhanced viral replication described above, causing a high level of viremia. The blood-brain-barrier flooded with virus particles, fails to act as a filter to virus invading the brain. In other words, the number of virions in the plasma by itself facilitates neuroinvasion. Our results demonstrate a non significant rise in viremia level in coldstressed mice. Moreover, when the SVN virus is injected intravenously in a twofold higher concentration, with subsequent higher viremia t l t e r , there is no brain invasion. Adding the fact that infection with neuroinvasive strains of Sindbis virus does not cause a higher viremia titer (S. LUSTIG, M. HALEVY, D. BEN-NATHAN, Y. AKOV - submitted for publication), we negate this second theory. The final theory suggests a stress induced blood-brain-barrler breakdown due to the release of agents that can change the blood-brain-barrler permeability. This breakdown is needed in addition to a sufficient level of viremia to make virus brain invasion possible. One should remember that brain invasion of as few as 10 PFU or less can cause encephalitis. Thus, a minor blood-brain-barrier breach, permitting but a few virions invasion, is enough for causing encephalitis.
Vol. 48, No. 15, 1991
Stress-Induced Viral Brain Invasion
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Non neuroinvasive virus can penetrate into the brain following induced temporary blood-brain-battler breach that can be achieved by chemicals, or by breathing 27% C02 (24). Table 3 presents convincing results that a noninvasive virus can invade the brain following a mild brain trauma, giving additional confirmation to this theory. The agents that induce blood-brain-barrier breakdown have not been discovered yet, a theory concerning 5-HT has been mentioned (12). Other endogenous mediators which can fit in this theory are macrophage derived cytokines which are known to be involved in infections of the central nervous system (25) and are capable of increasing capillariy permeability (26). Mast cell constituents, known to be released during stress, can fit in this theory as well (27). Remembering that viruses themselves act as stressors, inducing the same endocrinological as well as immunomodulating changes as stress (28,29), we propose that viruses able of inducing such agents release, gain the trait of neuroinvasiveness. Studies searching fop such agents are in progress. The cold stress effect on the blood-brain-barrier can be explained by an additional direct mechanism. Cold injury, inducing stimulation of the ornithine decarboxilase enzyme in the brain, can lead to a blood-brain-barrier breakdown and subsequent entrance of molecules that cannot enter the brain otherwise (30). Isolation did not cause a decline in body temperature, thus rejecting this mechanism as a common mechanism for brain invasion. Our results provide the suggestion that there are no changes in SVN viral traits in stress exposed mice, and a stress induced blood-brain-barrier breach is most probably responsible for SVN neuroinvasion. The neurovirulent non-neuroinvasive viruses can serve as all or none markers for blood-brain-barrier permeability and aid in studying pathological processes of the brain. Moreover, we suggest the use of these viruses as viable pathogenic indicators for stress in the animal. Further investigations are warranted into the mechanism by which the bloodbrain-barrier is opened and into the link between stress and the entrance of other microorganisms as well as non viable molecules to the brain
References
I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16.
R. ADER, Psychoneuroimmunology. Academic Press. New York. (1981). V. RILEY, Science 2121100-1109 (1981). R. DANTZER, K.W. KELLEY, Life sci. 4_441995-2008 (1989). O. KANDILL, M. BORYSENKO, Brain Behav. Immun. 2 3 2 - 4 9 (1988). S.B. FRIEDMAN, L. GLASGOW, R. ADIR, Psychosom. Med. 3_22285-299 (1970). M.M. JENSEN, A.F. RASMUSSEN, J. Immunol. 9 0 2 1 - 2 3 (1963). M.P. ROGERS, M.D. DUBEY, P. REICH, Psychosom. Med. 4 1 1 4 7 - 1 6 4 (1979) A.F. RASMUSSEN, J.T. MARCH, Proc. Soc. Exp. Biol. Med. 96 183-189 (1957). S.S. CHANG, and A.F. RASMUSSEN, Nature. 205 623-625 (1965). D. BEN-NATHAN, and G. FEURSTEIN, Experientia. 46 285-290 (1990). A. GANNICLIFFE, R.N.P. SUTTON, and R.F. ITZHAKI, Psychol. Med. 16 247249 (1986). H.S. SHARMA, and P.K. DEY, Neurosci. Res. 5 224-239 (1988). T. BELOVA, Biull. EXp. Biol. Med. 108 101-105 (1989). D. BEN-NATHAN, S. LUSTIG, and G. FEURSTEIN, Arch. Virol. 109 110(1989). D.E. GRIFFIN, R.T. and JOHNSON, J. Immunol. 118 1070-1075 (1977). D.E.GRIFFIN, C.S. HAHN, A.C. JACKSON, S. LUSTIG, E.G. STRAUSS,J.H. and
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17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
Stress-Induced Viral Brain Invasion
Vol. 48, No. 15, 1991
STRAUSS, in: Cell bioloKy of virus entry, replication, and pathoKenesis. Alan R. Liss, Inc. 387-396. (1989). L.G. REED, and M. MUENCH, Am. J. HyE. 2_/7493-7 (1938). R. DULBEC0, and M. VOGT, J. Exp. Med. 99 167-182 (1956). G. MAESTRONI, A. CONTI, and W. PIERRAPOLI, Immunology. 63 465-469 (1988). B.S. RABIN, M. LYTE, L.H. EPSTEIN, and A.R. CAGGIULA, Ann. NY. Acad. Sci. 496 492-500 (1986). J.J. JESSOP,K. GALE, and B.M. BAYER, Life Sci. 43 1133-40 (1988). J. WEINBERG, and J.T. EMERMAN, Brain Behav. Immun. _3 234-246 (1989). R.T. JOHNSON, Viral infections of the brain. Raven press. (1982). D. KOBILER, S. LUSTIG, Y. GOZES, D. BEN-NATHAN, and Y. AKOV, Brain Research. 496 314-316 (1989). G.E. GRAU, P.F. PIGUET, P. VASSALI, and P.H. LAMBERT, Int. Arch. Allergy Appl. Immunol. 88 34-39 (1989). S.E. GOLDBLUM, B. HENNIG, M. JAY, K. YONEDA and C.I. McCLAIN, Inf. Immun. 57 1218-1226 (i989). T.C. THEOCHARIDES, Life Sci. 46 607-617 (1990). A.J. DUNN, M.L. POWELL, C. MEITHIN, and P.A. SMALL, Brain Behav. Immun. ! 216-30 (1987). A.J. DUNN, M.L. POWELL, W.V. MORESHEAD, J.M. GASKIN,and N.R. HALL, Physiol. Behav. 45 591-594 (1989). H. KOENIG, A.D. GOLDSTONE, and C.Y. LU, J. Neurochem. 52 101-109 (1989).
"
Pergamon Press
STRESS-INDUCED NEUROINVASIVENESS OF A NEUROVIRULENT NONINVASIVE SINDBIS VIRUS IN COLD OR ISOLATION SUBJECTED MICE D. B e n - N a t h a n Dept.
S. L u s t i g
of Virology, Israel Institute for P.O.Box 19, 70450 Ness-Ziona, (Received
in final
form February
H.D.
Danenberg
Biological Israel 8,
Research
1991)
Suw~ar 7 Effects of cold or isolation stress on brain penetration by the neurovirulent noninvasive Sindbis virus strain (SVN) w e r e s t u d i e d i n m i c e . SVN i n j e c t e d intracerebrally (i.c.) causes acute encephalitis and kills adult mice but is unable to invade the brain and kill when injected intraperitoneally (i.p.). Mice inoculated i . p . w i t h SVN w e r e e x p o s e d t o c o l d s t r e s s o r were singly housed. Both stress patterns induced SVN encephalitis and death in 42% (cold) and 37% (isolation) of the tested mice. No death was observed in the control injected miceg Brain virus levels were found to be more than i0- PFU in all dying mice. No virus was detected in the control group brains. The virus that was isolated from the brains of moribund mice demonstrated no changes in neuroinvasive and neurovirulent properties. We suggest a stress induced blood-brain-barrier opening with subsequent virus entrance as the mechanism of stress induced SVN encephalitis. Stress is a term defining the reaction of the body to a variety of emotional and physical stimuli which endanger homeostasis. Studies have demonstrated the intimate connections between stress and the immune system (1,2,3). The stress effects on the immune system in human and animal studies are most commonly suppressive, with changing production and activity of a wide range of immune system components. Viruses are known to flourish in the stressed host, which suffers increased morbidity and mortality (4,5,6,7,8). Studies concerning neurovirulent viruses report of increased morbidity and mortality as a consequence of stress treatment (9,10). As a rule, it is believed that at times of immunosuppression there is an increased rate of viral infection of the central nervous system. This phenomenon is explained either by persistance and reactivation of the virus in the brain or increased multiplication in the periphery of a virus with neuroinvasive properties (ii). Another mechanism
which
can
be
responsible
for
the
0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
increased
rate
of
1494
Stress-Induced Vira~ Brain Invasion
Vol. 48, No. 15, 1991
encephalitis is the disruption of the blood-brain-barrier and subsequent virus penetration. Blood-brain-barrier permeability to [131]-Iodlne-sodium was found increased at times of stress (12). Histological study of immobilized rat brains revealed microvascular damage in the midbrain (13). West Nile Virus-25 (WN-25) is an attenuated nonneuroinvaslve strain of West Nile Virus that does not produce any detectable viremla. Previous work studying the effect of stress on mice inoculated with WN-25, demonstrated changes in viral traits, resulting in the recovery from the brain of virus with neuroinvasive properties comparable to the neurolnvasive wild type WNV (14). Those remarkable results did neither reject or support the theory of stressinduced encephalitis due to blood-brain-barrier breach. The present study was planned to check the influence of stress on brain invasion by a noninvasive neurovirulent virus, that produces a high viremia titer and is not derived of a neuroinvasive virus. Sindbis virus neuroadapted strain (SVN) is an alphavirus not invading the brain in mice when injected intraperitoneally (i.p.), but causing encephalitis when injected intracerebrally (i.c.), even when in a very low dose (15,16 and S. LUSTIG, M. HALEVY, D. BEN-NATHAN, Y. AKOV - submitted for publication). Mice inoculated i.p. with SVN were subjected to various kinds of stress and checked for changes in mortality and virus levels in blood and brain tissue as well as other stress parameters. Materials and Methods Sindbls virus - neuroadapted strain (SVN) A Sindbis virus (SV) was isolated in 1983 from a pool of culicine mosquitoes gathered in southern Israel. It was lethal to suckling mice by all routes of injection, but without ill effects to weanling mice (outbred, ICR). This SV strain was passed consecutively by i.c. injection in suckling mice. A new passage variant (SVN) was isolated, which caused death in weanling mice in~ected i.c., but not in those injected i.p. Stock v~rus contained 7.5xi0 plaque forming units (PFU\ml). The i.c. titer was 3.4xi0" MICLD50kml. The stock virus was kept at -70°C. Mice Charles river mice CrI:CDR-I (ICR) female were obtained at the age of 21 days (10-12 g body weight) and kept in our vivarium until the age of 27 to 42 days. In all studies, mice of the same age and batch were used. Mice were divided into groups following inoculation and were observed for mortality until the end of the experiment (21 days). For virus level in the brain and for lymphoid organs weight, mice were sacrificed 7 days after inoculation. Virus inoculation Each mouse was inoculated i.p. with 1.5x105 PFU of SVN. The virus dilutions were performed using inactivated rabbit serum (10%) in 0.9% saline containing penicillin(l,000~/ml). Groups of 6-10 mice were used, and the results were calculated according to the method of Reed and Muench (17). Stress Cold stress Mice were placed for 5 minutes in 3 cm deep ice cold water (I±0.5°C). Mice were exposed to cold stress either twice, on inoculation day and the day after, or five times until 4 days post inoculation. Isolatlon Mice were housed in individual cages immediately after inoculation until the end of the experiment. Control mlce were housed 6 per cage. Veto cells The Veto cell line was derived from kidneys of normal African Green Monkeys. The cells were grown in Dulbeco's Modified Eagle Medium (DMEM) containing 10% fetal calf serum (FCS).
Vol. 48, No. 15, 1991
Stress-Induced Viral Brain Invasion
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Isolation of SVN from the brain of infected mice Each brain was rinsed in cold PBS and sonlcated, The virus suspension was centrifuged at 3,000 rpm for I0 min. The supernatant was alllquoted into plastic tubes and stored at -70°C until further processes. The virus level in the brain was determined by titration of virus in Veto cell line. Isolation of SVN from blood Cold stressed and control mice were bled at various time points from the tail vein into special test tubes (brand serum separator, Beckton Dickinson). Virus content in the serum was plaque-assayed in Veto cells. Organs weight Mice were individually weighed 7 days after inoculation. The mice were sacrificed and the thymus and spleen were removed immediately and weighed aseptically. Titration of virus in tissue cultures For demonstration of SVN plaques in Veto cells the original plaque technique of Dulbeco and Vogt (18) was used. A dilution of virus was added to the Vero cell monolayers in petri dishes and incubated at 37°C for lh to permit viral adsorption. The monolayer is overlaid with MEM x 2 and tragacanth (Gum tragacanth Grade III G-I128, Sigma) containing 2~ FCS and 2.4~ NaHC0 R. The cultures were incubated (37°C,5~C02) for 72h. Plaques were counted ~fter staining the monolayer with neutral red (0.05~). The same procedure was followed for BHK cells. All plaques were counted by an experienced investigator. In vlvo assay The virus isolated from the brain of dying mice was grown in Veto cells and injected i.p. or i.c. to non stressed mice. Mice were observed for mortality for a period of 21 days. Data analysis Results are expressed as means±SEM. The data were analyzed by one way ANOVA or Fisher exact probability test. Results The effect of stress on body temperature. Body temperature of mice exposed to cold water declined sharply to a temperature of 14°C and returned to a normal level about an hour and a half later. Results are shown in figure i. Isolation stress had no effect on body temperature compared to the control group. The effect of cold or isolation on body and lymphatic organ weight. To study the general effect of stress on mice, spleen and thymus weights were measured 7 days after SVN inoculation and exposure to stress. Isolation and cold stress both reduced spleen and thymus weight. SVN inoculation of non stressed mice induced a non significant reduction of lymphoid organs weight. Inoculation of stressed mice with SVN induced a significant weight reduction. The results are presented in figure 2. The e f f e c t of cold or isolation stress patterns on mortality of mice inoculated with SVN. These experiments were done to determine the effect of cold or isolation stress on mortality of mice after inoculation wi~h SVN. All groups except the control i.c. group were inoculated i.p. with 2x10- PFU of SVN. Results are presented in table I. The virus was proved to kill non-stressed mice only by the i.c. route, no mortality was observed in the control group that was injected i.p.. Cold stress, either two or five exposures, induced a mortality rate of 20% (2 exposures) to 42~ (5 exposures). The mortality rate of the single housed mice was 37~ (p<0.05 in all groups compared to control group). Death in all stress groups occurred on days 7,8,9 following inoculation with no significant difference in the mean time of death.
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Stress-Induced Viral Brain Invasion
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Vol. 48, No. 15, 1991
~
7
35 30 m
o
E=
D
4 , 3
15 2
10 oz
÷
5
25 20
t--
spllen/B.W.
~h~ thymus/B.W.
1
5 II,],IIIIL,I,I,I,
II
i,i,i,i,i
;3 -'0 30 40 50 60 70 80 9 0 1 0 0 1 1 0 ~20
240
conlrol
SVN
Time after exposure to cold water (rain)
cold
treatment
cold
+ SVN
Isolation
Isolation
÷SVN
group
FIGURE 2
FIGURE 1
Body temperature of mice exposed to cold water (n=9)
Ratio of lymphoid organ to body weight (xlO00) 2<0.05 TABLE
1
The effect of cold or isolation stress on mortality of SVN inoculated mice.
Treatment group
a
Mortality
D/T Control i.p. Control i.c 5 Cold 2 days b Cold 5 days Isolation
0/24 24/24 4/20 11/26 14/38
% 0 i00 20~ 42~ 37-
a
All groups, except the i.c. group, were injected i.p. with 2x105 PFU of SVN, control i.c. group were injected i0 PFU (0.03ml) of SVN. b - 5min at I±0.5°C immediately after inoculation, and on the day after (2d), and on the 4 consecutive days (5d). c - R<0.05 d - 2<0.01 D/T - Dead/Total. Virus level in the blood of stressed and non stressed mice Figure 3 illustrates virus level in the blood of cold-stressed and in nonstressed mice. Virus levels reached a peak at 24h post inoculation and than declined with no virus in the blood at 72h post inoculaion. The virus level in the stress group was less than a log higher than the level in the control group. Virus level on brain 7 days after inoculation and exposure to stress To determine brain virus level in stressed infected mice, brains were taken from moribund mice on day 7 after inoculation. As a control, brains were taken from non stressed infected mice. The results are presented in table 2. No virus was detected in brains of non-stressed injected mice, while in brains of moribund mice a significant virus titer was found.
Vol. 48, No. 15, 1991
Stress-Induced Viral Brain Invasion
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Inoculation of mice with the virus recovered from brains of isolation or cold stressed m i c e . In order5to study changes in neurolnvaslveness, 24 mlce were inoculated i.p. with 5xlO of virus isolated from brains of stressed mice (12 - isolation and 12 - cold ). 21 days of observation for mortality recorded no death. Brains taken from 6 mice at day 7 following inoculation were free of virus. Intracerebral inoculation of mice with the isolated virus had similar results to the original SVN virus.
Cold
•
-
SVN
+
0
-
SVN
Control
;
,
T
1
2
3
-
?
Days post inoculation
FIGURE 3 Viremia level in cold stressed mice (n=6)
TABLE 3
TABLE 2 The e f f e c t o f c o l d o r I s o l a t l o n on b r a i n SVN l e v e l a t 7 d a y s a f t e r i n o c u l a t i o n and e x p o s u r e t o s t r e s s .
The effect of i.c. PBS injection on mortality of mice inoculated peripherally w l t h SVN.
Treatment group
N
log10 PFU/brain
Treatment group
Control i.p. Control i°c. Cold (5 days) Isolation
8 6 8 8
~2 6.89±0.45 6.51±0.31 6.73±0.27
All groups except the isC. group were injected l.p. with 2xlO PFU of SVN. i.c. group were injected with i0 PFU. p
Mortality D/T
PBS SVN SVN SVN
i.c. only~ i.p.(5xlO-pfu) i.c.(lO pfu) i.p.+PBS l.c.
018 0116 818 14/16
0 0 I00 87.5
0.03ml of PBS injected i.c. 24h after inoculation.
I n j e c t i o n o f s a l i n e s o l u t i o n t o t h e b r a i n o f m i c e i n o c u l a t e d w i t h SVN. Table 3 presents the effect of mild mechanical trauma on the penetration of noninvaslve virus to the brain. Intracerebral injectlon'of O.03ml of PBS to mice 24h post inoculation with SVN (time of peak viremla) induced encephalitis and mortality in 87.5% of the mice. Injection of PBS to noninfected mice did not cause any mortality.
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Discussion The present study demonstrates an encephalitis inducing effect of stress on mice inoculated with a neurovirulent virus with no neuroinvasive properties. The stress patterns were designed to simulate two major kinds of stress, psychological and physical. While the psychological i.e. isolation stress, was of prolonged duration, the physical cold stress was of short term, applied either twice or several times. The results clearly exhibit similarity in stress effect on lymphoid organ weight, a common parameter to the general immunosuppressive properties of stress. The effect is attributed to elevated corticosterone levels and is known to be augmented in viral infection (19). As well as for lymphoid organ weight, the results clearly exhibit similarity in SVN encephalitis rate and mortality in the different stress exposed groups. Thus, suggesting a common mechanism of influence activated by various stress patterns which differ in type and duration. Single housing effects on the immune system are controversial, both increased (20,21) and decreased (2,22) susceptibility to disease were observed in mouse and rat models. The changes in the effect are most probably time-dependent with an immunosuppressive effect changing with time to immunoenhancement. Our previous experience with short-term isolation stress in mice is clearly in favor of an increased infection due to an immunosuppressive effect (i0,14). There are three known pathways of virus spread to the brain - hematogenous, neural, and via the olfactory nerve (23). The hematogenous spread is the one commonly taken by alphaviruses. The mechanism by which stress induces Sindbis virus neuroinvasiveness is not clear. However, it can be explained by three major different hypotheses. Suppressed immmune response in stress, with defective function of macrophages and lymphocytes, leads to enhancement of viral replication in the blood and peripheral organs, induction of a selection process, and selection of a neuroinvasive strain. This hypothesis seems valid in the case of the neuroinvaslveness change of the attenuated WNV strain (14). In the present study the virus recovered from brains of stress-subjected mice did not show any changes in the neuroinvasive properties, thus 8nnuling this theory. The second theory is also based on the enhanced viral replication described above, causing a high level of viremia. The blood-brain-barrier flooded with virus particles, fails to act as a filter to virus invading the brain. In other words, the number of virions in the plasma by itself facilitates neuroinvasion. Our results demonstrate a non significant rise in viremia level in coldstressed mice. Moreover, when the SVN virus is injected intravenously in a twofold higher concentration, with subsequent higher viremia t l t e r , there is no brain invasion. Adding the fact that infection with neuroinvasive strains of Sindbis virus does not cause a higher viremia titer (S. LUSTIG, M. HALEVY, D. BEN-NATHAN, Y. AKOV - submitted for publication), we negate this second theory. The final theory suggests a stress induced blood-brain-barrler breakdown due to the release of agents that can change the blood-brain-barrler permeability. This breakdown is needed in addition to a sufficient level of viremia to make virus brain invasion possible. One should remember that brain invasion of as few as 10 PFU or less can cause encephalitis. Thus, a minor blood-brain-barrier breach, permitting but a few virions invasion, is enough for causing encephalitis.
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Stress-Induced Viral Brain Invasion
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Non neuroinvasive virus can penetrate into the brain following induced temporary blood-brain-battler breach that can be achieved by chemicals, or by breathing 27% C02 (24). Table 3 presents convincing results that a noninvasive virus can invade the brain following a mild brain trauma, giving additional confirmation to this theory. The agents that induce blood-brain-barrier breakdown have not been discovered yet, a theory concerning 5-HT has been mentioned (12). Other endogenous mediators which can fit in this theory are macrophage derived cytokines which are known to be involved in infections of the central nervous system (25) and are capable of increasing capillariy permeability (26). Mast cell constituents, known to be released during stress, can fit in this theory as well (27). Remembering that viruses themselves act as stressors, inducing the same endocrinological as well as immunomodulating changes as stress (28,29), we propose that viruses able of inducing such agents release, gain the trait of neuroinvasiveness. Studies searching fop such agents are in progress. The cold stress effect on the blood-brain-barrier can be explained by an additional direct mechanism. Cold injury, inducing stimulation of the ornithine decarboxilase enzyme in the brain, can lead to a blood-brain-barrier breakdown and subsequent entrance of molecules that cannot enter the brain otherwise (30). Isolation did not cause a decline in body temperature, thus rejecting this mechanism as a common mechanism for brain invasion. Our results provide the suggestion that there are no changes in SVN viral traits in stress exposed mice, and a stress induced blood-brain-barrier breach is most probably responsible for SVN neuroinvasion. The neurovirulent non-neuroinvasive viruses can serve as all or none markers for blood-brain-barrier permeability and aid in studying pathological processes of the brain. Moreover, we suggest the use of these viruses as viable pathogenic indicators for stress in the animal. Further investigations are warranted into the mechanism by which the bloodbrain-barrier is opened and into the link between stress and the entrance of other microorganisms as well as non viable molecules to the brain
References
I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16.
R. ADER, Psychoneuroimmunology. Academic Press. New York. (1981). V. RILEY, Science 2121100-1109 (1981). R. DANTZER, K.W. KELLEY, Life sci. 4_441995-2008 (1989). O. KANDILL, M. BORYSENKO, Brain Behav. Immun. 2 3 2 - 4 9 (1988). S.B. FRIEDMAN, L. GLASGOW, R. ADIR, Psychosom. Med. 3_22285-299 (1970). M.M. JENSEN, A.F. RASMUSSEN, J. Immunol. 9 0 2 1 - 2 3 (1963). M.P. ROGERS, M.D. DUBEY, P. REICH, Psychosom. Med. 4 1 1 4 7 - 1 6 4 (1979) A.F. RASMUSSEN, J.T. MARCH, Proc. Soc. Exp. Biol. Med. 96 183-189 (1957). S.S. CHANG, and A.F. RASMUSSEN, Nature. 205 623-625 (1965). D. BEN-NATHAN, and G. FEURSTEIN, Experientia. 46 285-290 (1990). A. GANNICLIFFE, R.N.P. SUTTON, and R.F. ITZHAKI, Psychol. Med. 16 247249 (1986). H.S. SHARMA, and P.K. DEY, Neurosci. Res. 5 224-239 (1988). T. BELOVA, Biull. EXp. Biol. Med. 108 101-105 (1989). D. BEN-NATHAN, S. LUSTIG, and G. FEURSTEIN, Arch. Virol. 109 110(1989). D.E. GRIFFIN, R.T. and JOHNSON, J. Immunol. 118 1070-1075 (1977). D.E.GRIFFIN, C.S. HAHN, A.C. JACKSON, S. LUSTIG, E.G. STRAUSS,J.H. and
1500
17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
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Vol. 48, No. 15, 1991
STRAUSS, in: Cell bioloKy of virus entry, replication, and pathoKenesis. Alan R. Liss, Inc. 387-396. (1989). L.G. REED, and M. MUENCH, Am. J. HyE. 2_/7493-7 (1938). R. DULBEC0, and M. VOGT, J. Exp. Med. 99 167-182 (1956). G. MAESTRONI, A. CONTI, and W. PIERRAPOLI, Immunology. 63 465-469 (1988). B.S. RABIN, M. LYTE, L.H. EPSTEIN, and A.R. CAGGIULA, Ann. NY. Acad. Sci. 496 492-500 (1986). J.J. JESSOP,K. GALE, and B.M. BAYER, Life Sci. 43 1133-40 (1988). J. WEINBERG, and J.T. EMERMAN, Brain Behav. Immun. _3 234-246 (1989). R.T. JOHNSON, Viral infections of the brain. Raven press. (1982). D. KOBILER, S. LUSTIG, Y. GOZES, D. BEN-NATHAN, and Y. AKOV, Brain Research. 496 314-316 (1989). G.E. GRAU, P.F. PIGUET, P. VASSALI, and P.H. LAMBERT, Int. Arch. Allergy Appl. Immunol. 88 34-39 (1989). S.E. GOLDBLUM, B. HENNIG, M. JAY, K. YONEDA and C.I. McCLAIN, Inf. Immun. 57 1218-1226 (i989). T.C. THEOCHARIDES, Life Sci. 46 607-617 (1990). A.J. DUNN, M.L. POWELL, C. MEITHIN, and P.A. SMALL, Brain Behav. Immun. ! 216-30 (1987). A.J. DUNN, M.L. POWELL, W.V. MORESHEAD, J.M. GASKIN,and N.R. HALL, Physiol. Behav. 45 591-594 (1989). H. KOENIG, A.D. GOLDSTONE, and C.Y. LU, J. Neurochem. 52 101-109 (1989).