Vet. Pathol. 12: 387-393 (1975)
Mouse Encephalitis Induced by Avirulent Semliki Forest Virus M A YCHEW-LIM St. Thomas’ Hospital Medical School, London
Abstract. Encephalitis caused by avirulent Semliki Forest virus was studied at regular intervals for 6 weeks in experimentally infected mice. The most obvious lesion was perivascular cuffing. Microglia were predominant near the vessels early in the infection. The cells of the cuffs were mostly lymphocytes. Although the virus produced acute encephalitis, which gradually resolved by 6 weeks, at no time were there abnormal neurological clinical signs. The cells of the brain and the immune response reacted to eliminate the virus.
The first isolation of the Semliki Forest virus in 1944 from mosquitoes (Aedes sp.) in Uganda was by inoculation into mice [ll]. It was originally classified as an arthropod-borne virus [2] but is now in the Toga virus group [4]. The virions are 20-30 pm in size. The histology of encephalitis in mice protected by antiserum for the acute stage of the virulent strain of the virus has been described [lo]. Encephalopathy was reported in nine mice, 26 months after intracerebral inoculation with avirulent Semliki Forest virus [16]. A brief comment on brain reaction after intraperitoneal infection was also given [161. The present study traces the course of brain reaction after a single intraperitoneal inoculation of the avirulent Semliki Forest virus ; evaluates the different cell types that participate in such an inflammatory reaction ; and discusses the role of these cells in eliminating this virus, which produces changes in the central nervous system without clinical signs.
Materials and Methods The Semliki Forest virus was obtained from Dr. C. J. BRADISH (Microbiological Research Establishment, Porton, Salisbury). This avirulent A774 strain was originally isolated
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Table I. Degree of perivascular cuffing in brains of infected mice Days after inoculation
Grading of perivascular cuffs
7 9 I1 14 18 24 28 42
+ + and ++
++
U+
++++ U+
++ + in one brain out of four
from Aedes in Portuguese East Africa [7]. It had undergone seven intracerebral passages in 2- to 5-day-old mice and was a clonal selection from plaques of the virus when grown on monolayers of primary chicken embryo cells [I]. The dose used was 0.1 ml of 10/4 dilution of virus in a suspension of sterile bovine albumin phosphate saline at pH 7.0. Mice of the Swiss A2G strain 3 - 4 weeks old were used. They were of either sex from a statistically randomly bred colony maintained within a specific pathogen-free barrier. Twenty mice were given 0.1 ml of the virus intraperitoneally, and 10 controls were given 0.1 ml bovine albumin phosphate. They were killed on days 1-4, 7, 1 1 , 14, 18, 28, and 42 after inoculation. In a related experiment 20 infected mice and 20 controls were killed on days 7,9, 14, 24, and 42. Mouse brains were fixed in 10% buffered formol-saline for at least 7 days. Each brain was divided in the frontal plane into three parts. The most anterior cut was through the telencephalon about 2 mm anterior to the pineal body, in front of the hippocampus. The second cut was through the mesencephalon-metencephalon junction posterior to the hippocampus. This separated the cerebellum and medulla from the rest of the brain. Tissues were embedded in paraffin, cut at 5-6 pm and stained with hematoxylin and eosin (HE). The method of WEILand DAVENPORT [I41 modified by MARSHALL [ 5 ] was used for demonstration of microglia and oligodendrocytes in the paraffin sections. Frozen sections, 10 pm thick, of hippocampus were specifically stained to show astrocytes by modified Cajal’s gold sublimate method [15]. Perivascular cuffs were graded + to *+ on a basis similar to that used by WEBB et a/. [I 31. When the cuffs were incomplete in surrounding a blood vessel, these were considered as +. Grading was ++++ when the cuff contained several layers of cells.
Results
No abnormality was seen histologically on days 1, 2, 3 and 4. On day 7 all parts of the brain showed various degrees of a viral encephalitis graded
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2 Fig. 1. Section of hippocampus showing different microglia forms on day 1 1 . WeilDavenport. Fig. 2. Section of telencephalon showing perivascular cuff predominantly of lymphocytes on day 18. HE.
as +. Perivascular cuffing was composed largely of lymphocytes with rare plasma cells. On day 9 the cuffs were + and ++. The inflammatory perivascular response on day 11 was distinct and uniform in all parts of the brain and was graded as ++. Maximum perivascular cuff reaction graded +++ and ++++ was on day 14, 18, and 24 (table I). Perivascular cuffs were more obvious and numerous in the medullary substance of the cerebellum later on in the infection. On days 28 and 42 there were fewer cuffs, the reaction was much less, and grading was ++ or +. By day 42 three out of four brains examined showed no signs of encephalitis, and the one brain with perivascular cuffing had it in the medullary substance of the cerebellum. The cells identified as microglia in the infected brain varied from cells with short bodies and branching processes to elongated cells with few processes (rod cells). Intermediate forms between these two types and also the typical swollen histiocytic cells with eccentric nuclei and abundant cytoplasm were seen. Lamellae of microglia were around blood vessels. Microglial reaction was found not to be related to the intensity of perivascular cuffing. A very good microglial reaction was seen on days 7,9, l l , and 14 (fig. 1); however, when the perivascular cuffs were at their height on
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Fig. 3. Section of hippocampus showing hypertrophied astrocytes on day 42. Cajal’s gold sublimate.
day 18, most of the cells around and in the cuffs were identified as lymphocytes and plasma cells (fig. 2). Some microglial reaction was still identified in the molecular layer of the cerebellum on day 28, but many of the cells near the cuffs were identified as oligodendrocytes. The use of gold sublimate permitted recognition of the degree of astrocyte activity. In the earlier phases of the disease this was not conspicuous. Along with the cuffing, astrocytic hypertrophy and hyperplasia were most marked on days 14, 18, and 24. A few hypertrophied astrocytes (fig. 3) could still be seen on day 42 in the one brain positive for perivascular cuffs. There was no evidence of neuronal damage.
Discussion The criterion for viral encephalitis was cellular activity in the brain consisting of perivascular cuffing, microglial proliferation, and astrocytic reaction. In using the perivascular cuff to monitor the brain reaction it was found that the first cuff noticed on day 7 coincided with a brain virus titre of 105.2the day the brain titre began to fall (as compared to the titre of 10s.5 on
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day 4) [8]. When serum antibody was first detected on day 9 [8], the perivascular cuffing was still increasing. On day l l , when brain virus was only just detectable at a titre of [8], perivascular cuffs were much more distinct. When encephalitis was severe and extensive on days 14, 18, and 24, no virus was recovered from the brain [8]. On days 28 and 42 cuffing was diminished, and by day 42 only one brain out of four showed perivascular cuffs. The function of microglia in the reactions described could be to phagocytose the virus and products of virus activity in an attempt to stop virus from infiltrating into the brain substance. The transport of virulent Semliki Forest virus from the blood vessel into the central nervous system of baby mice has been reported, and virus particles were seen in microglia-like cells [9]. It is also possible that microglia, as part of the lymphoreticular system, play a role in processing of the virus. The perivascular cuff is an important part of the immune response. DOHERTY et al. [3], in experiments with the louping-ill virus given subcutaneously to sheep, demonstrated by immunofluorescent technique specific antilouping-ill IgG in the plasma cells of the perivascular cuff of the surviving sheep. Fibre-forming astrocytes are seen where there is massive destruction of white matter. The hypertrophied fibrous cells have an important role in cicatrix formation to replace damaged cerebral tissue. Astrocyte activity was related to perivascular cuffs, and hypertrophied astrocytes could still be seen on day 42 in the brain with cuffs. In these mice it was a reaction to the virus infection. It cannot be compared directly to the findings of ZLOTNIK [15], in whose experiments the virulent strain of Semliki Forest virus was used; however, the astrocyte picture is not dissimilar to that in mice killed 26 months after intracerebral inoculation with avirulent virus [16]. The other cells identified were oligodendrocytes, which seemed to be more numerous in the white matter of the cerebellum near the perivascular cuffs on day 28. This apparent increase could be the result of an attempt to replace any small amount of myelin lost because of the virus infection. The two experiments illustrate the cell types involved in an intraperitoneal infection with an avirulent strain of a neurotropic virus. The central nervous system may be the seat of a severe encephalitis, without clinical neurological signs such as paralysis being seen. Killing the mice at regular intervals showed that by 4-6 weeks the brain had successfully resolved any encephalitis detectable by microscopic examination. It is difficult to compare the present findings with those of ZLOTNIKet al. [16] in 1972, as the author made no
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progressive analysis of brain changes up to the point of 26 months. Subsequently I examined single brains 8 months and 14 months after intraperitoneal inoculation with the same strain of the virus and found no lesions of the central nervous system. Even in the experimental transmission of the spongiform encephalopathies to primates there was evidence that some progression of lesions preceded the onset of clinical signs, which took an average incubation period of 12 months [ 6 ] . The mouse brain has obviously been able to deal with avirulent Semliki Forest virus adequately. The histological picture is a simple one and related directly to the particular avirulent strain. The brain reaction to viruses varies between species and even among individuals in any sample of infected animals. Ultimately, it is the definitive host that copes with the virus. Even though no abnormal clinical signs were seen in these experimental mice, there was a transient reduction in spontaneous motor activity of approximately 5096, as measured by the Animex activity meter when brain virus titres were high [12].
A cknou~ledgenients The author thanks Dr. R.O. BARNARD for his advice and J.D. BRIGHTfor technical assistance. Supported in part by a grant from the Philip Fleming Trust.
References 1 BRADISH, C. J.; ALLNER, K., and MABER,H.B.: The virulence of original and derived strains of Semliki Forest virus for mice, guinea pigs and rabbits. J. gen. Virol. 12:
141-160 (1971). 2 CASALS, J. and BROWN,L.V.: Hemagglutination with arthropod-borne viruses. J. exp. Med. 99: 429-449 (1 954). 3 DOHERTY, P.C.; REID,H.W., and SMITH,W.: Louping-ill encephalomyelitis in the sheep. IV. Nature of the perivascular inflammatory reaction. J. comp. Path. Ther. 81: 545-549 (1971). 4 HORZINEK, M.: Comparative aspects of Toga viruses. J. gen. Virol. 20: 87-103 (1973). 5 MARSHALL, A.H.E.: An outline of the cytology and pathology of the reticular tissue, pp. 258-260 (Oliver & Boyd, London 1956). 6 MASTERS, C. L.; KAKULES, B. A.; GAJDUSEK, D. C., and GIBLS, C. J. : Subclinical lesions and their progression in the experimental spongiform, encephalopathies, Kuru and Jakob-Creutzfeldt disease in primates. 7th Int. Congr. of Neuropathology, Budapest 1974.
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7 MCKINTOSH, B.M.; BROOKWORTH, C., and KOKERNOT, R.H.: Isolation of Semliki
Forest virus from Aedes (Aedimorphus) argenteopunctatus (Theobald) collected in Portuguese East Africa. Trans. R. SOC.trop. Med. Hyg. 55: 192-198 (1961). 8 OATEN,S. and WEBB,H. E. : Personal commun. 9 PATHAK, S. and WEBB,H.E.: Possible mechanisms for the transport of Semliki Forest virus into and within mouse brain. An electron-microscopic study. J. neurol. Sci. 23: 175-184 (1974). 10 SEAMER, J. H. ; BOULTER, E. A., and ZLOTNIK, I. : Delayed onset of encephalitis in mice passively immunised against Semliki Forest virus. Br. J. exp. Path. 52: 408414 (1971). 11 SMITHBURN, K. C. and HADDOW, A. J. : Semliki Forest virus. I. Isolation and pathogenic properties. J. Immun. 49: 141-157 (1944). 12 SUCKLING, A. J.; BATEMAN, S., and WEBB,H.E.: A new method for monitoring subclinical virus infections of the central nervous system in mice. Br. J. exp. Path. 56: 287-290 (1975). 13 WEBB,H.E.; WIGHT,D. G. D.; PLATT,G. S.,and SMITH,C. E. G. : Langat encephalitis in mice: the effect of the administration of specific antiserum. J. Hyg., Camb. 66: 343354 (1968). H. A. : Staining of oligodendroglia and microglia in celloidin 14 WEIL,A. and DAVENPORT, sections. Archs Neurol. Psychiat. 30: 175-178 (1933). 15 ZLOTNIK, I.: The reaction of astrocytes to acute virus infections of the central nervous system. Br. J. exp. Path. 49: 555-564 (1968). 16 ZLOTNIK, I.; GRANT, D. P., and DEE-BATTER-HATTON: Encephalopathy in mice following inapparent Semliki Forest virus infection. Br. J. exp. Path. 53: 125-129 (1972).
Dr. MAYG.B. LIM,St. Thomas’ Hospital Medical School, London, SE1 7EH (England)