C E N T R A L N E R V O U S SY ST E M A L T E R A T I O N S A S S E Q U E L A E O F V E N E Z U E L A N E Q U I N E E N C E P H A L I T I S V I R U S INF E C T I O N I N THE R A T AND JosB ESPARZA~ JORGEGARC~A-TAMAYO*, GABRIEL CARREGOT
*Institute Anatomopatoldgico, Facultad de Medicina, Universidad Central de Venezuela and t Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela
PLATES LI-LVI
V E N E Z U E L Aequine N encephalitis (VEE) virus is lethal to the majority of rodents and other laboratory animals (Victor, Smith and Pollack, 1956; Gleiser et al., 1962). The pathogenic action of this virus is characterised by necrotising lesions in the central nervous system (CNS) and lymphohaematopoietic organs (Victor et al., Gleiser et al.; Gorelkin, 1972; Dill, Pederson and Stookey, 1973). The finding that VEE virus may produce nonlethal infections when administered in moderate doses to wild rodents has raised the possibility of latent infections in reservoirs (Young, Johnson and Gauld, 1969). However, very little is known regarding either the morphological changes occurring during sublethal infection or the CNS lesions resulting as sequelae produced after VEE virus infection. This type of investigation is important because of the high morbidity observed during VEE epidemics (Ryder, 1972). Moreover, a recent communication reported cerebral dysfunction in children who suffered from encephalitis caused by VEE virus (Leon et al., 1975). The aim of the present study was to examine, with the light and electron microscope, the CNS alterations in rats which had recovered from intraperitoneal (TP) inoculation with a virulent strain of VEE virus. MATERIALS AND METHODS Virus and virus assay The strain of VEE virus used in the present study (Goajira strain, IVIC-PH 117s) was originally isolated in 1962 from the serum of a patient during an epidemic in Northwestern Venezuela(SellersetaZ., 1965 ; Bergold and Mazzali, 1968). Virus was used after two passages in BHK-21 cells, followed by three passes in suckling mouse brain. Virus stocks were prepared in Vero cells, and infectivity titres calculated by a plaque method utilising an agarose serum-free overlay (Bergold and Mazzali). Animals and Experimental design FiftymaleSprague-Dawleyratsapproximately100 days old were used in these experiments. Forty rats were inoculated intraperitoneally with 200 plaque-forming units of VEE virus Acceptedfor publication 25 Sept. 1978. Address reprint request to: Dr Jorge Garcia-Tamayo, Instituto Anatomopatol6gico-UCV, Apartado 50647, Caracas, Venezuela. J. PATH.-VOL.
128 (1979)
87
88
GARCIA-TAMAYO,
CARREGOAND
ESPARZA
contained in 0.2 ml. Ten rats were kept as uninoculated controls. Animals were carefully checked for clinical manifestations of disease (fever, irritability, malaise and paralysis). Special care was taken to assure the recovery of inoculated animals, such as a quiet dark room and individual feeding and forced oral hydration during the critical period of the acute infection. Virological studies showed that the peak of viraemia occurred 2 days after infection, but the maximum amount of virus in the brain was detected during rhe 6th day of infection. Despite the special care taken, 25 rats died between 7 and 15 days after inoculation. Survivors were apparently recovered when examined 22 days after inoculation. Animals were sacrificed 1 or 3 months after infection. Histopathology
Brains were carefully removed and fixed in 10 per cent. formalin, dehydrated, embedded in paraffin, sectioned and stained with haematoxylin and eosin (H&E). Electron microscopy
Small fragments of cerebral cortex, basal ganglia, mesencephalon and cerebellum were fixed in 2.5 per cent. phosphate buffered glutaraldehyde, pH 7.2, at 4°C. Samples were washed in the buffer, post-fixed in 2 per cent. osmium tetroxide for 2 hr, dehydrated in alcohol and embedded in Araldite. Thin sections were obtained with a diamond knife-equipped Reichert ultramicrotome, and stained with uranyl acetate and lead citrate. Sections were examined in a Zeiss 9A electron microscope.
RESULTS Light microscopy The CNS of rats examined 1 mth after IP inoculation with VEE virus showed foci of cavitary necrosis in the cerebral cortex with macrophagic Gitter cells and mononuclear cell infiltrates in the peripheral vessels (fig. 1). Mononuclear cell infiltrates were also observed in the meninges and surrounding blood vessels in the cerebral cortex, hypocampus and mesencephalon. There was moderate astrocytic proliferation in the vicinity of cavitary lesions in the cortex. Microglial cells were increased in number in the cerebral grey matter, particularly in areas where the neuronal population was decreased. Perivascular cuffing with mononuclear cells and focal reduction in number of pyramidal neurons were noted in the hypocampus. Three months after IP inoculation there were some macrophagic Gitter cells in the areas of cavitary necrosis and many blood vessels with peripheral astrocytic fibres (figs, 2, 3 and 4). Mononuclear cells and microglial rods were observed in the vicinity of the cavitary lesions in the cerebral cortex. Cuffing of blood vessels with mononuclear cells was not so prominent as in the brain of rats examined 1 mth after IP inoculation. Microglial response was diffuse and more conspicuous in focal areas of the cerebral cortex. Small focci of spongy degeneration in the white matter with some microglial rods and mononuclear cells were also noted. Electron microscopy Electron microscopic findings were characterised by the presence of mononuclear round or amoeboid cells infiltrating the nervous tissue.
PLATELI FIG.1.-Mononuclear FIG.2.-Scarred FIG.3.-Detail FIG.4.-Area
cell infiltrates in meninges and cerebral cortex, one month after VEE virus infection. Haematoxylin and eosin (H&E). x 400.
area in the cerebral cortex 3 months after VEE virus infection. H&E.
x 100.
of fig. 2. Cortical necrosis with astrogliosis and microglial activity. H&E. ~ 4 4 0 . of cavitary necrosis in the cerebral cortex 3 mth after VEE virus infection. H&E. x 440.
GARC~A-TAMAYO, CARRERO
AND
PLATE LI
ESPARZA
EQUINE ENCEPHALITIS
FIG.1
FIG.2
FIG.3
FIG.4
GARCIA-TAMAYO, CARREGO AND ESPARZA
PLATE LII
EQUINE ENCEPHALITIS
FIG.5.-Mononuclear
FIG.6.-Mononuclear
cell in the lumen of a blood vessel. Ameboid cells and a plasmocyte in the perivascular space. x 4000.
ameboid cells extending their pseudopods into the neuropil.
~4000.
GARC~A-TAMAYO, CARRERO AND ESPARZA
PLATE LIII
EQUINE ENCEPHALITIS
FIG. 7. -Pseudopods of macrophages becoming adherent to nerve cell. x 15,000.
G A R C f A - T A M A Y O , C A R R E f i O AND
PLATE LIV
E,SPARZA EQUINE ENCEPHALITIS
FIG.8
FIG.9
FIG. 8. -Slender ameboid pseudopods o f a macrophage dissecting the neuropil. x 15,000. FIG.9.-Giant
swollen astrocyte with abundant glial fibrils. x 4000.
GARCIA-TAMAYO, CARREGO AND
PLATE LV
ESPARZA
EQUINE ENCEPHALITIS
FIG.10.-Endothelial
cell with the basal membrane and periciytic mononuclear ameboid cells.
Fic. 11.-Swollen
astrocyte in the neuropil.
~4000.
,’
15,000.
GARCiA-TAMAYO, C A R R E 3 0 AND
PLATE LVI
ESPARZA
EQUINE ENCEPHALITIS
FIG.13
FIG.12 FIG.12.-Degenerative FIG.13.-Detail
axonal changes. A non-myelinated area along a swollen axon is shown.
x 4000.
of a swollen axon with dense bodies, abundant mitochondria and filaments.
x 15,000.
EQUINE ENCEPHALITIS
89
The perivascular spaces revealed small lymphocytes, macrophages and some plasma cells (fig. 5). Mononuclear macrophagic amoeboid cells seemed to correspond to microglia and appeared to migrate away from the perivascular areas in the neuropil (fig. 6). Apericytic origin of the microglial cells seemed evident in some areas while in others the macrophagic amoeboid cells seemed to originate from the blood (fig. 10). The nucleus was oval or indented and there were lysosomes in the cytoplasm. The pseudopods of these cells were frequently slender and they penetrated and dissected the neuropil and became adherent to the nerve cells surrounding them (figs. 7 and 8). In some of these neurons there was evidence of alterations in the cytoplasmic organellae with swelling of the cytoplasm and rupture of the membranes. The foot processes of the astrocytes were swollen and there were astrocytes enlarged with abundant glial fibrils (figs. 9 and 11). Some astrocytes also showed binucleation. Axonal changes were characterised by focal areas of swelling with abundant osmiophilic masses and whorls of membranous bodies and mitochondriae (figs. 12 and 13). Some longitudinally sectioned axons showed these changes in a focal and repeated sequence with a normal appearance of the myelin sheath (fig. 12).
DISCUSSION The mechanism by which VEE virus inflicts damage to the nervous tissue may be by inducing necrotic changes as the result of virus multiplication in the nerve cells. This seems to be the case in newborn mice and other laboratory rodents (Victor et af., 1956; Gorelkin, 1973). We are not aware of previous reports of CNS alterations in rodents or other laboratory animals after sublethal infections with VEE virus, Our findings of cavitary necrosis, macrophage activity and mononuclear cell infiltrates may correspond to the areas of necrosis and acute inflammatory changes observed during the first 10 days after inoculation. Even when in the acute stage of the infection the rats were severely ill, no clinical evidence of disease was observed in the surviving animals. Light and electron microscopic examination of necrotic areas in the CNS showed infiltrates with celIs apparently of haematogenous origin, namely monocytes, plasma cells and lymphocytes. These cells were perivascular and interstitial in distribution and they correspond to the cellular inflammatory reaction characteristic of most viral encephalitis (McFarland, Griffin and Johnson, 1972). The origin of the mononuclear cell infiltrates observed in the CNS during virus infection has been extensively discussed (Maxwell and Kruger, 1965; Fujita and Kitamura, 1957). Both haematogenous cells and pericytes can be transformed into cerebral macrophages and they have been named microglia, cerebral macrophages or " M cells (Maxwell and Kruger; Matthews and Kruger, 1973). Our electron microscopic findings were consistent with the macrophagic nature of these invading cells with pseudopods extending into the neuropil. Venezuelan Equine Encephalitis virus particles were not observed in the cytoplasm of macrophages, glial or nerve cells in rats surviving the acute infection. The presence of mononuclear cells and "
90
CARC~A-TAMA YO,
CARRESO AND
ESPARZA
macrophages could be interpreted as the cellular immune response against virus specific antigens in the membrane of nerve cells. The electron microscopic finding of axonal changes in the CNS of rats observed 3 mth after the VEE virus infection was suggestive of transynaptically degenerative alterations in the nerve cells. Degenerative changes in the neurons of mice and monkeys following inapparent Semliki Forest virus (SFV) infection or after recovery from clinical SFV encephalitis have been considered as sequelae of the acute infection (Zlotnik, Grant and Batter-Hatton, 1972). It is well known that neuropil processes of injured cells may be secondarily damaged by lysosomal enzymes (Cos, 1976) and there is electron microscopic evidence for the release of lysosomal enzymes during VEE virus infection in the brain of newborn mice (Garcia-Tamayo, 1971). It seems reasonable to assume that lysosomal enzymes play an important role in the development of necrosis and further persistence of the inflammatory reaction. Whether this cellular response is due to the direct effect of the virus on nerve cells or to an immune response to cellular targets, such as virus coded surface antigens is not clear. The absence of virus particles in nerve cells adds strength to the latter hypothesis. It was previously suggested that VEE virus may persist latently in infected rats (Young et ai., 1969) and that other arboviruses are capable of initiating a sub-acute degenerative disease in the brain of experimental animals (Zlotnik, Grant and Carter, 1976). In addition, mosquito cells infected with VEE virus can establish latent infections with the virus information kept repressed in an inducible form (Carrefio and Esparza, 1977). Histological and ultrastructural observations of severe damage in the CNS, mainly in the cerebral cortex, may be considered as sequelae of acute VEE virus infection. It is important to point out that mononuclear cell activity persisted in the CNS 3 mth after the acute onset of infection in rats which appear to be healthy. It is likely that these focal lesions may be the counterpart of similar lesions which may occur after VEE virus infection in humans, since epilepsy and other clinical evidence of cerebral dysfunction have been reported in children who suffered from encephalitis caused by VEE virus (Leon et al., 1975).
SUMMARY Alterations of the Central Nervous System (CNS) in rats surviving acute infection with a virulent strain of Venezuelan Equine Encephalitis (VEE) virus were studied by light and electron microscopy. Cavitary necrosis of the cerebral cortex, macrophage activity and degenerative axonal changes were considered to be sequelae of the lesions induced during the acute phase of the infection. Mononuclear cell infiltrates of the neuropil, 3 mth after inoculation, were related to the immune response of the host. Focal lesions and mononuclear cell activity in the brain are thought to be the equivalent of the lesions induced in the CNS of humans during VEE virus infection. The findings are discussed in the light of recent reports of cerebral dysfunction occurring as a sequel of VEE virus infection in children.
EQUINE ENCEPHALITIS
91
REFERENCES MAZZALI, R. 1968. Plaque formation by arboviruses. J . Gen. Virol.,
BERGOLD, G. H., AND 2,273-284. CARREGO, G., AND ESPARZA, J. 1977. Induction of Venezuelan Equine Encephalitis (Mucambo) virus by Iododeoxyuridine in chronically infected " cured " cultured mosquito cells. Zntervirology, 8, 193-203. COX,V. S. 1976. Ultrastructure of the axon reaction in the inmature rat thalamus. J . Neuropath. Exper. Neurol., 35, 191-203. DILL,G. S., PEDERSON, C. E., AND STOOKEY, J. L. 1973. A comparison of the tissue lesions produced in adult hamsters by two strains of avirulent Venezuelan Equine Encephalomyelitis virus. Am. J. Pathol., 72, 13-23. FUJITA,S., AND KITAMURA, T. 1975. Origin of brain macrophages and the nature of the so-called microglia. Acta Neuropath (Berlin) Suppl. VI, 291-296. GARC~A-TAMAYO, J. 1971. Acid phosphatase activity in mouse brain infected with Venezuelan Equine Encephalomyelitis virus. J. Virol., 8, 232-241. GLEISER, C. A., GOCHENOUR, W. S., BERGE, T. O., AND TIGERTT, W. D. 1962. The comparative pathology of experimental Venezuelan Equine Encephalomyelitis infection in different animals hosts. J. Infect. Dis., 110, 80-97. GORELKIN, L. 1973. Venezuelan Equine Encephalomyelitis in adult animal host. An electron microscopic study. Am. J. Pathol., 73, 425442. LEON,c. A., JARAMILLO, R., MARTINEZ, S., FERNANDEZ, F., TELLER, H., LASSO,B., AND DE GUZMAN, R. 1975. Sequelae of Venezuelan Equine Encephalitis in humans: a four-year follow-up. I n t . J . Epidemiol., 4, 131-140. MCFARLAND, H. F., GRIFFIN, D. E., AND JOHNSON, R. T. 1972. Specificity of the inflammatory reponse in viral encephalitis. J. Exp. Med. 136, 216-226. MATTHEWS, M. A., AND KRUGER, L. 1973. Electron microscopy of non-neuronal cellular changes accompanying neural degeneration in the thalamic nuclei of the rabbit. 11. Reactive elements within the neuropil. J. Comp. Neurol., 148, 313-346. MAXWELL, D. S., AND KRUGER, L. 1965. Small blood vessels and the origin of phagocytes in the rat cerebral cortex following heavy particles irradiation. Exp. Neurol., 12, 33-54. RYDER, S. 1972. Encefalitis Equina Venezolana. Aspectos epidemiologicos de la enfermedad entre 1962 y 1971, en la Guajira Venezolana. Invest. Clin., 13, 91-141. SELLERS, R. F., BERGOLD, G. H., SUAREZ, 0. M., AND MORALES, A. 1965. Investigations during Venezuelan Equine Encephalitis outbreaks in Venezuela 1962-1964. Amer. J. Trop. Med. Hyg., 14, 460-469. VICTOR, J., SMITH, D. G., AND POLLACK, A. D. 1956. The comparative pathology of Venezuelan Equine Encephalomeylitis. J . Infect. Dis.,98, 55-66. YOUNG,N. A., JOHNSON, K. M., AND GAULD,L. W. 1969. Viruses of the Venezuelan Equine Encephalomyelitis complex. Experimental infection of Panamanian rodents. Am. J. Trop. Med. Hyg., 18, 290-296. ZLOTNIK, I., GRANT, D. P., AND BATTER-HATTON, D. 1972. Encephalopathy in mice following inapparent Semliki forest virus (SFV) infection. Br. J . Exp. Pathol., 53, 125-129. ZLOTNIK, I., GRANT,D. P., AND CATER,G. B. 1976. Experimental infection of monkeys with viruses of the tick-borne encephalitis complex : degenerative cerebellar lesions following inapparent forms of the disease of recovery from clinical encephalitis. Br. J. Exp. Path., 57, 200-210.
*Institute Anatomopatoldgico, Facultad de Medicina, Universidad Central de Venezuela and t Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela
PLATES LI-LVI
V E N E Z U E L Aequine N encephalitis (VEE) virus is lethal to the majority of rodents and other laboratory animals (Victor, Smith and Pollack, 1956; Gleiser et al., 1962). The pathogenic action of this virus is characterised by necrotising lesions in the central nervous system (CNS) and lymphohaematopoietic organs (Victor et al., Gleiser et al.; Gorelkin, 1972; Dill, Pederson and Stookey, 1973). The finding that VEE virus may produce nonlethal infections when administered in moderate doses to wild rodents has raised the possibility of latent infections in reservoirs (Young, Johnson and Gauld, 1969). However, very little is known regarding either the morphological changes occurring during sublethal infection or the CNS lesions resulting as sequelae produced after VEE virus infection. This type of investigation is important because of the high morbidity observed during VEE epidemics (Ryder, 1972). Moreover, a recent communication reported cerebral dysfunction in children who suffered from encephalitis caused by VEE virus (Leon et al., 1975). The aim of the present study was to examine, with the light and electron microscope, the CNS alterations in rats which had recovered from intraperitoneal (TP) inoculation with a virulent strain of VEE virus. MATERIALS AND METHODS Virus and virus assay The strain of VEE virus used in the present study (Goajira strain, IVIC-PH 117s) was originally isolated in 1962 from the serum of a patient during an epidemic in Northwestern Venezuela(SellersetaZ., 1965 ; Bergold and Mazzali, 1968). Virus was used after two passages in BHK-21 cells, followed by three passes in suckling mouse brain. Virus stocks were prepared in Vero cells, and infectivity titres calculated by a plaque method utilising an agarose serum-free overlay (Bergold and Mazzali). Animals and Experimental design FiftymaleSprague-Dawleyratsapproximately100 days old were used in these experiments. Forty rats were inoculated intraperitoneally with 200 plaque-forming units of VEE virus Acceptedfor publication 25 Sept. 1978. Address reprint request to: Dr Jorge Garcia-Tamayo, Instituto Anatomopatol6gico-UCV, Apartado 50647, Caracas, Venezuela. J. PATH.-VOL.
128 (1979)
87
88
GARCIA-TAMAYO,
CARREGOAND
ESPARZA
contained in 0.2 ml. Ten rats were kept as uninoculated controls. Animals were carefully checked for clinical manifestations of disease (fever, irritability, malaise and paralysis). Special care was taken to assure the recovery of inoculated animals, such as a quiet dark room and individual feeding and forced oral hydration during the critical period of the acute infection. Virological studies showed that the peak of viraemia occurred 2 days after infection, but the maximum amount of virus in the brain was detected during rhe 6th day of infection. Despite the special care taken, 25 rats died between 7 and 15 days after inoculation. Survivors were apparently recovered when examined 22 days after inoculation. Animals were sacrificed 1 or 3 months after infection. Histopathology
Brains were carefully removed and fixed in 10 per cent. formalin, dehydrated, embedded in paraffin, sectioned and stained with haematoxylin and eosin (H&E). Electron microscopy
Small fragments of cerebral cortex, basal ganglia, mesencephalon and cerebellum were fixed in 2.5 per cent. phosphate buffered glutaraldehyde, pH 7.2, at 4°C. Samples were washed in the buffer, post-fixed in 2 per cent. osmium tetroxide for 2 hr, dehydrated in alcohol and embedded in Araldite. Thin sections were obtained with a diamond knife-equipped Reichert ultramicrotome, and stained with uranyl acetate and lead citrate. Sections were examined in a Zeiss 9A electron microscope.
RESULTS Light microscopy The CNS of rats examined 1 mth after IP inoculation with VEE virus showed foci of cavitary necrosis in the cerebral cortex with macrophagic Gitter cells and mononuclear cell infiltrates in the peripheral vessels (fig. 1). Mononuclear cell infiltrates were also observed in the meninges and surrounding blood vessels in the cerebral cortex, hypocampus and mesencephalon. There was moderate astrocytic proliferation in the vicinity of cavitary lesions in the cortex. Microglial cells were increased in number in the cerebral grey matter, particularly in areas where the neuronal population was decreased. Perivascular cuffing with mononuclear cells and focal reduction in number of pyramidal neurons were noted in the hypocampus. Three months after IP inoculation there were some macrophagic Gitter cells in the areas of cavitary necrosis and many blood vessels with peripheral astrocytic fibres (figs, 2, 3 and 4). Mononuclear cells and microglial rods were observed in the vicinity of the cavitary lesions in the cerebral cortex. Cuffing of blood vessels with mononuclear cells was not so prominent as in the brain of rats examined 1 mth after IP inoculation. Microglial response was diffuse and more conspicuous in focal areas of the cerebral cortex. Small focci of spongy degeneration in the white matter with some microglial rods and mononuclear cells were also noted. Electron microscopy Electron microscopic findings were characterised by the presence of mononuclear round or amoeboid cells infiltrating the nervous tissue.
PLATELI FIG.1.-Mononuclear FIG.2.-Scarred FIG.3.-Detail FIG.4.-Area
cell infiltrates in meninges and cerebral cortex, one month after VEE virus infection. Haematoxylin and eosin (H&E). x 400.
area in the cerebral cortex 3 months after VEE virus infection. H&E.
x 100.
of fig. 2. Cortical necrosis with astrogliosis and microglial activity. H&E. ~ 4 4 0 . of cavitary necrosis in the cerebral cortex 3 mth after VEE virus infection. H&E. x 440.
GARC~A-TAMAYO, CARRERO
AND
PLATE LI
ESPARZA
EQUINE ENCEPHALITIS
FIG.1
FIG.2
FIG.3
FIG.4
GARCIA-TAMAYO, CARREGO AND ESPARZA
PLATE LII
EQUINE ENCEPHALITIS
FIG.5.-Mononuclear
FIG.6.-Mononuclear
cell in the lumen of a blood vessel. Ameboid cells and a plasmocyte in the perivascular space. x 4000.
ameboid cells extending their pseudopods into the neuropil.
~4000.
GARC~A-TAMAYO, CARRERO AND ESPARZA
PLATE LIII
EQUINE ENCEPHALITIS
FIG. 7. -Pseudopods of macrophages becoming adherent to nerve cell. x 15,000.
G A R C f A - T A M A Y O , C A R R E f i O AND
PLATE LIV
E,SPARZA EQUINE ENCEPHALITIS
FIG.8
FIG.9
FIG. 8. -Slender ameboid pseudopods o f a macrophage dissecting the neuropil. x 15,000. FIG.9.-Giant
swollen astrocyte with abundant glial fibrils. x 4000.
GARCIA-TAMAYO, CARREGO AND
PLATE LV
ESPARZA
EQUINE ENCEPHALITIS
FIG.10.-Endothelial
cell with the basal membrane and periciytic mononuclear ameboid cells.
Fic. 11.-Swollen
astrocyte in the neuropil.
~4000.
,’
15,000.
GARCiA-TAMAYO, C A R R E 3 0 AND
PLATE LVI
ESPARZA
EQUINE ENCEPHALITIS
FIG.13
FIG.12 FIG.12.-Degenerative FIG.13.-Detail
axonal changes. A non-myelinated area along a swollen axon is shown.
x 4000.
of a swollen axon with dense bodies, abundant mitochondria and filaments.
x 15,000.
EQUINE ENCEPHALITIS
89
The perivascular spaces revealed small lymphocytes, macrophages and some plasma cells (fig. 5). Mononuclear macrophagic amoeboid cells seemed to correspond to microglia and appeared to migrate away from the perivascular areas in the neuropil (fig. 6). Apericytic origin of the microglial cells seemed evident in some areas while in others the macrophagic amoeboid cells seemed to originate from the blood (fig. 10). The nucleus was oval or indented and there were lysosomes in the cytoplasm. The pseudopods of these cells were frequently slender and they penetrated and dissected the neuropil and became adherent to the nerve cells surrounding them (figs. 7 and 8). In some of these neurons there was evidence of alterations in the cytoplasmic organellae with swelling of the cytoplasm and rupture of the membranes. The foot processes of the astrocytes were swollen and there were astrocytes enlarged with abundant glial fibrils (figs. 9 and 11). Some astrocytes also showed binucleation. Axonal changes were characterised by focal areas of swelling with abundant osmiophilic masses and whorls of membranous bodies and mitochondriae (figs. 12 and 13). Some longitudinally sectioned axons showed these changes in a focal and repeated sequence with a normal appearance of the myelin sheath (fig. 12).
DISCUSSION The mechanism by which VEE virus inflicts damage to the nervous tissue may be by inducing necrotic changes as the result of virus multiplication in the nerve cells. This seems to be the case in newborn mice and other laboratory rodents (Victor et af., 1956; Gorelkin, 1973). We are not aware of previous reports of CNS alterations in rodents or other laboratory animals after sublethal infections with VEE virus, Our findings of cavitary necrosis, macrophage activity and mononuclear cell infiltrates may correspond to the areas of necrosis and acute inflammatory changes observed during the first 10 days after inoculation. Even when in the acute stage of the infection the rats were severely ill, no clinical evidence of disease was observed in the surviving animals. Light and electron microscopic examination of necrotic areas in the CNS showed infiltrates with celIs apparently of haematogenous origin, namely monocytes, plasma cells and lymphocytes. These cells were perivascular and interstitial in distribution and they correspond to the cellular inflammatory reaction characteristic of most viral encephalitis (McFarland, Griffin and Johnson, 1972). The origin of the mononuclear cell infiltrates observed in the CNS during virus infection has been extensively discussed (Maxwell and Kruger, 1965; Fujita and Kitamura, 1957). Both haematogenous cells and pericytes can be transformed into cerebral macrophages and they have been named microglia, cerebral macrophages or " M cells (Maxwell and Kruger; Matthews and Kruger, 1973). Our electron microscopic findings were consistent with the macrophagic nature of these invading cells with pseudopods extending into the neuropil. Venezuelan Equine Encephalitis virus particles were not observed in the cytoplasm of macrophages, glial or nerve cells in rats surviving the acute infection. The presence of mononuclear cells and "
90
CARC~A-TAMA YO,
CARRESO AND
ESPARZA
macrophages could be interpreted as the cellular immune response against virus specific antigens in the membrane of nerve cells. The electron microscopic finding of axonal changes in the CNS of rats observed 3 mth after the VEE virus infection was suggestive of transynaptically degenerative alterations in the nerve cells. Degenerative changes in the neurons of mice and monkeys following inapparent Semliki Forest virus (SFV) infection or after recovery from clinical SFV encephalitis have been considered as sequelae of the acute infection (Zlotnik, Grant and Batter-Hatton, 1972). It is well known that neuropil processes of injured cells may be secondarily damaged by lysosomal enzymes (Cos, 1976) and there is electron microscopic evidence for the release of lysosomal enzymes during VEE virus infection in the brain of newborn mice (Garcia-Tamayo, 1971). It seems reasonable to assume that lysosomal enzymes play an important role in the development of necrosis and further persistence of the inflammatory reaction. Whether this cellular response is due to the direct effect of the virus on nerve cells or to an immune response to cellular targets, such as virus coded surface antigens is not clear. The absence of virus particles in nerve cells adds strength to the latter hypothesis. It was previously suggested that VEE virus may persist latently in infected rats (Young et ai., 1969) and that other arboviruses are capable of initiating a sub-acute degenerative disease in the brain of experimental animals (Zlotnik, Grant and Carter, 1976). In addition, mosquito cells infected with VEE virus can establish latent infections with the virus information kept repressed in an inducible form (Carrefio and Esparza, 1977). Histological and ultrastructural observations of severe damage in the CNS, mainly in the cerebral cortex, may be considered as sequelae of acute VEE virus infection. It is important to point out that mononuclear cell activity persisted in the CNS 3 mth after the acute onset of infection in rats which appear to be healthy. It is likely that these focal lesions may be the counterpart of similar lesions which may occur after VEE virus infection in humans, since epilepsy and other clinical evidence of cerebral dysfunction have been reported in children who suffered from encephalitis caused by VEE virus (Leon et al., 1975).
SUMMARY Alterations of the Central Nervous System (CNS) in rats surviving acute infection with a virulent strain of Venezuelan Equine Encephalitis (VEE) virus were studied by light and electron microscopy. Cavitary necrosis of the cerebral cortex, macrophage activity and degenerative axonal changes were considered to be sequelae of the lesions induced during the acute phase of the infection. Mononuclear cell infiltrates of the neuropil, 3 mth after inoculation, were related to the immune response of the host. Focal lesions and mononuclear cell activity in the brain are thought to be the equivalent of the lesions induced in the CNS of humans during VEE virus infection. The findings are discussed in the light of recent reports of cerebral dysfunction occurring as a sequel of VEE virus infection in children.
EQUINE ENCEPHALITIS
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REFERENCES MAZZALI, R. 1968. Plaque formation by arboviruses. J . Gen. Virol.,
BERGOLD, G. H., AND 2,273-284. CARREGO, G., AND ESPARZA, J. 1977. Induction of Venezuelan Equine Encephalitis (Mucambo) virus by Iododeoxyuridine in chronically infected " cured " cultured mosquito cells. Zntervirology, 8, 193-203. COX,V. S. 1976. Ultrastructure of the axon reaction in the inmature rat thalamus. J . Neuropath. Exper. Neurol., 35, 191-203. DILL,G. S., PEDERSON, C. E., AND STOOKEY, J. L. 1973. A comparison of the tissue lesions produced in adult hamsters by two strains of avirulent Venezuelan Equine Encephalomyelitis virus. Am. J. Pathol., 72, 13-23. FUJITA,S., AND KITAMURA, T. 1975. Origin of brain macrophages and the nature of the so-called microglia. Acta Neuropath (Berlin) Suppl. VI, 291-296. GARC~A-TAMAYO, J. 1971. Acid phosphatase activity in mouse brain infected with Venezuelan Equine Encephalomyelitis virus. J. Virol., 8, 232-241. GLEISER, C. A., GOCHENOUR, W. S., BERGE, T. O., AND TIGERTT, W. D. 1962. The comparative pathology of experimental Venezuelan Equine Encephalomyelitis infection in different animals hosts. J. Infect. Dis., 110, 80-97. GORELKIN, L. 1973. Venezuelan Equine Encephalomyelitis in adult animal host. An electron microscopic study. Am. J. Pathol., 73, 425442. LEON,c. A., JARAMILLO, R., MARTINEZ, S., FERNANDEZ, F., TELLER, H., LASSO,B., AND DE GUZMAN, R. 1975. Sequelae of Venezuelan Equine Encephalitis in humans: a four-year follow-up. I n t . J . Epidemiol., 4, 131-140. MCFARLAND, H. F., GRIFFIN, D. E., AND JOHNSON, R. T. 1972. Specificity of the inflammatory reponse in viral encephalitis. J. Exp. Med. 136, 216-226. MATTHEWS, M. A., AND KRUGER, L. 1973. Electron microscopy of non-neuronal cellular changes accompanying neural degeneration in the thalamic nuclei of the rabbit. 11. Reactive elements within the neuropil. J. Comp. Neurol., 148, 313-346. MAXWELL, D. S., AND KRUGER, L. 1965. Small blood vessels and the origin of phagocytes in the rat cerebral cortex following heavy particles irradiation. Exp. Neurol., 12, 33-54. RYDER, S. 1972. Encefalitis Equina Venezolana. Aspectos epidemiologicos de la enfermedad entre 1962 y 1971, en la Guajira Venezolana. Invest. Clin., 13, 91-141. SELLERS, R. F., BERGOLD, G. H., SUAREZ, 0. M., AND MORALES, A. 1965. Investigations during Venezuelan Equine Encephalitis outbreaks in Venezuela 1962-1964. Amer. J. Trop. Med. Hyg., 14, 460-469. VICTOR, J., SMITH, D. G., AND POLLACK, A. D. 1956. The comparative pathology of Venezuelan Equine Encephalomeylitis. J . Infect. Dis.,98, 55-66. YOUNG,N. A., JOHNSON, K. M., AND GAULD,L. W. 1969. Viruses of the Venezuelan Equine Encephalomyelitis complex. Experimental infection of Panamanian rodents. Am. J. Trop. Med. Hyg., 18, 290-296. ZLOTNIK, I., GRANT, D. P., AND BATTER-HATTON, D. 1972. Encephalopathy in mice following inapparent Semliki forest virus (SFV) infection. Br. J . Exp. Pathol., 53, 125-129. ZLOTNIK, I., GRANT,D. P., AND CATER,G. B. 1976. Experimental infection of monkeys with viruses of the tick-borne encephalitis complex : degenerative cerebellar lesions following inapparent forms of the disease of recovery from clinical encephalitis. Br. J. Exp. Path., 57, 200-210.
