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    Lesions in the cerebral cortexes of ponies inoculated with enzootic Venezuelan equine encephalomyelitis VEE IE NVSL 93-42124 virus isolate. A, Severe menengoencephalitis (arrow) in pony 3 (bar = 10 mm). B, Mild menengoencephalitis in pony 2, showing mostly infiltration of monocytes (arrow) (bar = 3.4 mm). C, Extensive perivascular cuffing in pony 1, showing infiltration of monocytes (arrows) (bar = 10 mm). D, Gliosis (arrow) and perivascular cuffing of a small blood vessel (arrowhead) in pony 1 (bar = 5 mm); E, Perivascular cuffing (rectangular area is shown in F) of a large blood vessel in pony 2 (bar = 10 mm). F, Enlargement of the rectangular area in E, showing mostly infiltration of lymphocytic cells (arrow) and endothelial cells lining of blood vessel (arrrowheads) (bar = 5 mm). G, Neuronal degeneration (arrows) and swollen glial cells (arrowheads) in pony 3 (bar = 5 mm). (Hematoxylin and eosin stained.)

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PATHOGENICITY OF A VENEZUELAN EQUINE ENCEPHALOMYELITIS SEROTYPE IE VIRUS ISOLATE FOR PONIES

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  • 1 U.S. Department of Agriculture, Animal and Plant Health Inspection Service, National Veterinary Services Laboratories, Ames, Iowa

The enzootic or endemic strains of Venezuelan equine encephalomyelitis (VEE) virus (ID, IE, IF, and II-VI) are considered avirulent. In 1993 and 1996, outbreaks of encephalitis occurred in the horse populations in the Chiapas and Oaxaca provinces of Mexico, respectively. In both instances, enzootic VEE virus subserotype IE was isolated from brain tissues of dead horses. The present study investigated the pathogenicity of the Chiapas viral isolate (NVSL VEE IE 93-42124) in ponies. Three ponies were inoculated intradermally with 4, 5, and 6 logs, respectively, of the NVSL VEE IE 93-42124 viral isolate. All ponies showed fluctuations in body temperature, encephalitis, and other signs of infection with VEE virus. Virus was isolated only from the blood of ponies from day 1 to day 3 postinfection. Microscopic examination of hematoxylin and eosin–stained tissue sections showed mild to moderate nonsuppurative encephalitis, perivascular cuffing by mononuclear cells, gliosis, and meningoencephalitis. Antibody (IgM) to VEE virus IE was unable to differentiate between various subserotypes of VEE I viruses (serotypes IAB, IC, ID, and IF). Virus neutralizing antibody titers to heterologous VEE I viruses were 10–100-fold less than those for NVSL VEE IE 93-42124 virus and Mena II, a human isolate of VEE IE virus. The study confirmed that NVSL VEE IE 93-42124 virus, which was isolated from a brain of a horse during an outbreak of VEE in Chiapas, Mexico, was pathogenic for ponies.

INTRODUCTION

The Venezuelan equine encephalomyelitis (VEE) virus is an enveloped, positive-strand RNA virus of the family Togaviridae, genus Alphavirus and is divided antigenically into several serotypes: IAB to IF and II to VI.1 The VEE virus is transmitted through the bite of mosquitoes and can cause acute illness in equines, humans, and other species.1 Based on pathogenicity to equines and humans, the VEE virus complex is divided into two major groups: an epizootic or epidemic form and an enzootic or endemic form.1 The epizootic or epidemic form of VEE viruses (IAB and IC) can cause high mortality and morbidity in equines and humans.1–5 Equines serve as amplifiers of the epizootic form of the viruses; thus, they become the source of infection of thousands of mosquitoes during the febrile period.1,2 The epizootic viruses affect various tissues of viscera and the central nervous system (CNS). Pathologic lesions of naturally occurring epizootic VEE IAB virus infections in horses and humans have been reported, as have experimental infections of horses, donkeys, monkeys (Macaca mulatta), rodents, and bats with virulent VEE IAB virus.6–14

Histopathologic lesions were observed in multiple organs and tissues, especially the brain, gastrointestinal tract, lungs, spleen, and lymph nodes, in 21 autopsied human patients who died of documented VEE during the 1962–1963 epidemic in Zulia, Venezuela. In the CNS, mild or focal mixed inflammatory cell infiltration was present in the leptomeninges and perivascular spaces. Meningoencephalitis associated with intense necrotizing vacuities was also observed. However, there was a striking depletion of lymphocytes with vascular thrombosis and necrosis of follicles in lymph nodes, spleen, and gastrointestinal tract. Widespread hepatocellular degeneration and individual cell necrosis and interstitial pneumonia were observed.7 Monkeys infected with epizootic VEE IAB virus showed no clinical disease except a biphasic febrile response. Lesions in brain tissues, lymphatic tissues, and liver were similar to those observed in humans. Lesions were also observed in the kidneys and heart.

The cerebral cortexes from 15 horses that died of naturally occurring VEE virus infection in Carmelo and other epizootic foci in Colombia10 showed perivascular inflammatory reactions with lymphocytes and neutrophils. Both astrocytes and neutrophils were found in the perineuronal spaces of the cerebral cortex. Focal glial nodules, satellitosis, and lymphocytic perivascular reactions were often seen in the medulla and cerebellum. In experimental studies, one fatal case showed severe pathologic changes in the bone marrow, spleen, lymph nodes, pancreas, liver, and kidneys.6 No significant lesions were seen in other visceral organs. Horses that died of encephalitis had marked vascular changes in the brain tissues that were not limited to any particular area of the brain.6 Pathologic changes consisted of swollen endothelial cells, perivascular edema and cuffing, infiltration of polymorpho-nuclear leucocytes, and slight neuron damage. Perivascular cuffing with lymphocytes and mononuclear cells was seen 8–20 weeks after infection in the cerebral cortexes of the horses that survived. However, severe lesions in the brains were observed in the cerebral cortexes of dead, moribund, and recovered horses infected with VEE IAB virus.11,15,16 Experimental cases of donkeys inoculated with virulent VEE IAB virus had similar pathologic lesions of VEE virus as those observed in the brain, lymph nodes, spleen, pancreas, liver, and kidneys of experimentally infected horses.6,7,11,13 In the brain, the majority of the lesions were in the cerebral cortex, and there were few lesions in the pons, thalamus, and hippocampus.

Large brown bats (Eptesicus fuscus) were found to be resistant to VEE IAB virus isolates.12 Despite the multiplication of VEE virus in the blood, liver, spleen, and brain of bats, with levels approaching those demonstrable in the brain tissues of mice succumbing to infection with the virus, no overt signs of the disease were observed in any of infected bats. Examination of hematoxylin and eosin–stained sections of brain tissues from bats when brain virus titers were highest (at 6 days postinfection) showed no evidence of pathology compared with sections of brain tissues from moribund infected mice that showed gliosis in brain tissues. These mice also had viral neutralizing antibodies in serum.

The enzootic or endemic forms (ID, IE, IF, and II-VI) of VEE virus are considered avirulent for horses and seldom cause overt disease in humans and animals.1,2,17,18 The main reason for their nonepidemic behavior is their lack of virulence for horses. Rodents and wild birds are natural hosts for enzootic VEE viruses and Culex mosquito species are the principal vectors.17–22 Culex taeniopus, a mosquito commonly found in Central America (from Mexico to Panama), has been shown to transmit VEE IE virus, but appears to be refractory to infection by the IAB and IC viral serotypes.18–22 Culex taeniopus and C. ocossa are known to be active disseminators of enzootic VEE IE virus.23 As early as 1960, reports of sickness and death of horses showing signs similar to that of VEE viral infections were observed in the states of Tamaulipas and Veracruz on the eastern coast of Mexico.2,24 More than 1,000 of 66,227 Mexican horses were affected by this disease and the mortality rate was 30%.24 In another area, 12% of 60 horses were affected and 4.3% died. This outbreak of the disease coincided with an increase in populations of mosquitoes, rodents, and birds, and occurred prior to the introduction of an epizootic strain of VEE virus type IAB.1,2,20,24,25 No virus isolate was available for serologic identification.

In 1963, a VEE virus identified as 63U2 was isolated from a sentinel hamster in Veracruz, Mexico.20 When the hemagglutination-inhibiting antigen of VEE virus 63U2 was tested against horse sera collected in Veracruz, Mexico during the 1960 outbreak, titers of 1:10 to 1:80 were obtained, indicating an outbreak of VEE.24 Serologic cross-reaction studies using homologous antiserum to this virus and antisera to previously known VEE isolates from Trinidad, Colombia, Panama, and the vaccine strain TC-83 indicated that isolate 63U2 was different from previously known VEE viral isolates.17 In addition, 16 VEE viruses isolated between 1963 and 1965 in Veracruz, Mexico were shown to be serologically similar to that of VEE Mena-II, a human isolate belonging to serogroup IE.17 Experimental infection of horses with serotype IE Mena-II failed to induce signs of the disease, although low concentrations of virus were present in their blood for a few days after infection.2 The isolation of VEE viruses from 1965 to 1968 in countries south of Mexico, including Guatemala, Honduras, and Belize, indicated that these viruses were similar to the 63U2 virus isolate and belonged to the IE subsero-group.21 These viruses were isolated in swamps (wet forests or lowlands of the Atlantic and the Pacific habitats). In experimental studies, eight different isolates of VEE virus serotype IE were inoculated into horses.15,16 Three isolates, 63R20, an isolate from sentinel mouse brain in Veracruz, Mexico, and 2177B and Nic. 2199, both isolated in 1968 from sentinel hamster brains in Nicaragua, were fatal to horses. The inoculated horses showed signs of infection with VEE virus, and low levels of virus was isolated from the blood and cerebrospinal fluid. In case of isolate 2177B, seven horses inoculated intradermally did not show signs of infection with VEE virus or death. However when three horses were inoculated intrathecally (inoculation through the spinal cord into the subarachnoid space) with isolate 2177B, two of them died and low amounts of virus were isolated from brain tissues. The investigators concluded that the death of the horses was due to causes other than VEE IE viral infection. It appeared that their conclusions were based on the earlier belief that VEE IE serogroup viruses were nonpathogenic.2

During the wet season of 1993, an outbreak of VEE occurred on the Pacific Coast in the state of Chiapas in Mexico.26 Chiapas is the southernmost state of Mexico, bordering Guatemala, and is a known habitat of the endemic subserogroup IE of VEE virus. In this outbreak, approximately 125 of 417 horses were affected and 50% of the cases were fatal. All affected horses showed clinical signs of typical VEE virus infection including fever, anorexia, ataxia, and walking in circles. Brain, lung, liver, and spleen tissues samples from four dead horses were sent to the National Veterinary Services Laboratories in Ames, Iowa for virus isolation and identification of the encephalitis agent(s). Venezuelan equine encephalomyelitis virus NVSL 93-42124 was isolated from the brain tissue of a horse and was identified as subtype IE (Table 1).26 In July 1996, a second outbreak of VEE occurred in the adjacent state of Oaxaca, Mexico. Thirty-two horses were affected and 12 died. Venezuelan equine encephalomyelitis virus NVSL 96-32863 was isolated from brain samples of two dead horses, and these isolates were identified as VEE subtype IE (96-32863-Oax 131 and Oax 142).26 Genetic characterization (nucleotide sequence of the 26S RNA regions) of various isolates of VEE viral subtype IE showed that the Chiapas (NVSL 93-42124) and Oaxaca (NVSL 96-32863) VEE viral IE isolates were similar, but 1.5% divergent from IE 68U201, the 1968 Hamster isolate from Guatemala, and 7% divergent from IE Mena II, the human isolate from Panama.26 The prototype VEE virus IE Mena II was isolated in 1962 from the blood of a human patient with a febrile reaction and CNS symptoms.2,17,18 Venezuelan equine encephalitis IE viruses were isolated from rodents, wild birds, and mosquitoes from Panama, Guatemala, Honduras, and Mexico and were antigenically indistinguishable from the Mena II human isolate.17–22 Venezuelan equine encephalitis virus subserotype IE was observed to be infectious and pathogenic to humans.21,24,27

Antibodies to the VEE IE virus in endemic areas in Central America were found to be widespread among the human population.21 Four cases of the infection in humans in Mexico were reported.27 Two were moderately severe, one naturally acquired was encephalitic and fatal, and one acquired by aerosol exposure in the laboratory. The disease was established by virus isolation and an increase in antibodies titers to VEE virus IE 63-U2, a Mexican strain.27 These findings indicate that humans as well as horses are susceptible to VEE IE virus. However, during 1993 and 1996 outbreaks of VEE in horses, no human cases were reported.26 It is possible that the infections in humans were mild in nature and not recognized as infections with VEE virus. The lack of efficient transmitters of VEE virus to humans may have also been a factor. In addition, no serologic test for the presence of the antibodies to the prevailing VEE IE virus isolate was available to establish human infection.

The purpose of the present study was to evaluate the pathogenicity of the 1993 Chiapas VEE isolate, IE NVSL 93-42124, in ponies by evaluation of their serum antibody responses.

MATERIALS AND METHODS

Ponies and housing.

Three ponies negative for antibodies to eastern, western, and Venezuelan equine encephalitis viruses were used in this study. The ponies were housed in a biosafety level 3 facility.28 Biosafety level 3 practices were followed by all individuals associated with this project.28 The project was reviewed and approved by the scientific committee of National Veterinary Services laboratories.

Virus.

The VEE virus isolate IE NVSL 93-42124 was passaged once in Vero (green monkey kidney) cells and the number of plaque-forming units (PFU)/mL was determined in Vero cells.2,26 The stock virus was frozen at −70°C before use. The isolate was identified as subserotype IE by a monoclonal antibody test (Table 1), a plaque-reduction neutralization test (PRNT), and genetic analysis.26

Infection of the ponies.

Prior to the start of the project, all personnel associated with this project were vaccinated with killed VEE viral vaccine. Each individual had a serum neutralizing titer > 1:32. Each pony was housed individually for 10 days before the start of the experiment. To establish a baseline for normal behavior of the ponies to facilitate identification of clinical signs of VEE virus infection and disease, the following parameters were noted: rectal temperature, eating and drinking habits, gait and posture, sensitivity to sound and light, any eye problems, depression, responsiveness to the command of an animal handler, and hind and foreleg coordination to evaluate the degree of weakness, ataxia, stiffness, and crossing or overreaching steps. One milliliter of viral inoculum was inoculated intradermally. The first, second, and third pony were inoculated with 1 × 104 (4 logs), 1 × 105 (5 logs), and 1 × 106 (6 logs) of VEE IE virus NVSL 93-42124, respectively.

Clinical signs and necropsy findings.

Each pony was observed for signs of infection with VEE virus. Signs of VEE included undulant fever, anorexia, nasal discharge, tears lacrimation scleritis, restlessness, constipation, and CNS signs. The CNS signs included mild to severe depression, yawing, hypersensitivity to sound and bright light, aimless chewing, walking and circling, goose-stepping, foot stomping, droopy ears, staggering, and ataxia.2

The ponies were humanely killed when they were judged to have an irreversible nervous disorder. A 5-mL sample of blood was collected into tubes containing EDTA for virus isolation and an additional 10 mL of blood was collected into tubes without anticoagulant for serologic analysis. These were collected daily from each pony. Necropsies were performed on all three ponies and paired samples of brain, lung, liver, kidney, lymph nodes, and spleen were taken aseptically for virus isolation and histopathologic examination. Proper treatment of laboratory animals are reviewed by members on Animal Care and Use Protocol. They follow the regulations of the US department of Agriculture written in Code of Federal Regulations (CFR) #9.

Mouse ascitic fluid and monoclonal antibodies.

Mouse ascitic fluid (MAF) to VEE virus TC-83 (serosubtype IAB) was used as a polyclonal antibody control and MAF to uninfected mouse brain was used as a control. Mouse ascitic fluid was produced as previously described.29 Briefly, equal volumes of Freundt’s adjuvant and killed antigen of VEE TC-83 virus (titer = 1 × 109 PFU/ml) in a suckling mouse brain sample were mixed, and 0.2 ml of this mixture was inoculated intraperitoneally into five 8-week-old mice. The process was repeated every week for three weeks. On the fourth week, all mice were given 0.1 ml of live VEE TC-83 virus (1 × 106 PFU) intraperitoneally. On the fifth week, mice were inoculated intrapertoneally with actively growing sarcoma 180 cells. After 10–20 days, MAF was harvested by aspiration from the peritoneal cavity of mice anesthetized with CO2. A similar method was used to produce normal MAF, except that uninfected suckling mouse brain was used in place of brain infected with VEE TC-83 virus.

A panel of monoclonal antibodies was obtained in 1992 from Dr. Nick Karabatsos (Centers for Disease Control and Prevention, Fort Collins, CO). Information about specificity of various monoclonal antibodies in various serologic tests (indirect fluorescent antibody test [IFA], enzyme-linked immunosorbent assay [ELISA], neutralization test, hemagglutination-inhibition test) for different serotypes and subserotypes of alpha viruses was obtained from the same source. Monoclonal antibodies were selected and tested for specificity to various VEE I AB, IC, ID, IE, and IF subserotype viruses by an IFA test (Table 1). The specificities of the monoclonal antibodies for various alpha viruses have been previously reported.30–33

Antiserum to VEE TC-83 (subserotype IAB).

Antiserum to VEE TC-83 was previously produced in a pony. Briefly, a pony was inoculated intradermally with 1 × 107 virus particles, and blood was collected on various days after inoculation. Sera were frozen at −20°C. The monospecificity of IgM antibodies to VEE I viruses is not known. Serum from the pony infected with TC-83 virus was included in the study to see if IgM antibody to VEE subserotype IE virus showed a mono-specific reaction, a one-way cross-reaction, or a cross-reaction with all viruses of VEE I serotypes.

Virus isolation and identification.

All samples for virus isolation were frozen at −70°C until tested. One gram of each tissue sample was homogenized in 10 ml of tissue culture medium with antibiotics (Gentamicin sulfate, (Boehringer Ingelhim, St. Joseph, MO) 1 ml/liter of the media), centrifuged in an enclosed container at 3,000 × g for one hour at 4°C, and supernatant was used for virus isolation. All blood and tissue supernatants were diluted in Eagle’s minimal essential medium (MEM) with antibiotics. Homogenized brain sample was a mixture of pons, cerebellum, cerebrum, meninges, thalamus, hippocampus, choroids plexus, and medulla of the spinal cord. Blood and tissue homogenate samples (undiluted, 1:5, 1:10, and 1:100) were used for virus isolation. One milliliter of each dilution of the samples was added to three 25-cm2 flasks containing 72-hour-old Vero cells. The inocula were allowed to adsorb onto Vero cells for one hour at 37°C. The inoculum was then discarded from the each flask and 10 ml of MEM with 2% calf serum was added to each flask. The flasks were incubated for 5–6 days at 37°C and examined daily for a cytopathic effect (CPE). If no CPE was observed after six days, the samples were passaged two more times before declaring the samples negative for virus isolation.

A 1:10 dilution of the supernatant of the brain homogenates were made in 0.01 M phosphate-buffered saline (PBS), pH 7.8, containing 0.75% albumin from all ponies and 0.20 μL of each sample was inoculated intracerebrally into 3–5-day-old suckling mice. The inoculated mice were observed for 10 days. Re-isolation of virus was attempted from brain of dead suckling mice by passaging once into the Vero cells. Only brains homogenates were used to inoculate the mice. Isolated virus was identified by an IFA test (Table 1) and a virus neutralization test.

IgM-capture ELISA.

The ELISA was performed as previously described.34 Briefly, all wells of ELISA microplates (Immulon-1B; Dynex Technologies, Inc., Chantilly, VA) were coated with a 1:400 dilution of anti-equine IgM in freshly prepared 0.5M carbonate buffer, pH 9.6. The plates were incubated at 4°C overnight in a humidified chamber. Before the start of the test, wells were blocked with 5% nonfat dry milk in 0.01M PBS containing 0.05% Tween 20 (PBS-T), pH 7.4, for one hour at 37°C. All sera were diluted in PBS-T from 1:100 to 1:100,000. Three negative, and one strongly positive, and one weakly positive sera were included as controls. Sera were tested in duplicate. Fifty microliters of the diluted sera samples were placed in predetermined wells of coated, blocked ELISA plates and incubated for 75 minutes at 37°C in a humidified chamber. Pretested, acetone-extracted VEE viral antigens from mouse brain and uninfected mouse brain antigens were added to the respective wells. The microplates were incubated overnight at 4°C in a humidified chamber. After incubation, the plates were washed with PBS-T and hydrogen peroxidase–conjugated monoclonal antibody to alphavirus (2A2C-3) was added to each well. The plates were incubated at 37°C for 75 minutes and substrate (ABTS) was added.35 The optical density (OD) was measured at 405 nm in a microplate reader. A serum dilution was considered positive for VEE viral antigens when the OD of the test serum was at least two times the mean OD value of a 1:100 dilution of the negative sera with respective viral antigens.

Plaque-reduction neutralization test.

A PRNT was used as previously described.2,26 Briefly, all sera were inactivated at 56°C for 30 minutes and then diluted in MEM as follows: 1:5, 1:10, 1:100, 1:1,000, 1:2,000, 1:4,000, 1:10,000, and 1:100,000. Each dilution of sera were tested with 100 PFU of TC-83, IE (NVSL 93-42124 and Mena-II), and IF VEE viruses. Virus-serum mixtures were incubated at 37°C for 60 minutes and then inoculated onto four-day-old Vero cells in 25-cm2 flasks. After adsorption for one hour at 37°C, 6 ml of agar overlay media was added to each flask. All flasks were incubated at 37°C for three days. The highest dilution resulting in a 90% reduction of virus plaques was considered the titer of the serum.

Histopathologic examination.

Postmortem tissue specimens (brain, lungs, liver, kidney, and spleen) were immersion fixed in 10% buffered formalin, processed according to standard procedures, and embedded in paraffin blocks. The brain tissues embedded for histopathologic examinations were the cerebellum, cerebrum, pons, thalmus, hipocampus, choroids plexus, and medulla, and spinal cord. Two to three different parts of tissues of each sample were embedded in paraffin blocks, and three thin sections were cut per block for histopathologic examinations. Histologic sections were cut at five microns, mounted on glass slides, and stained with hematoxylin and eosin. More than one block was examined only when samples appeared to have mild or no lesions.

RESULTS

Clinical disease.

The clinical signs of ponies inoculated with VEE IE virus isolate NVSL 93-42124 are shown in Table 2. All three ponies showed a fluctuating body temperature. The signs of CNS dysfunction were variable. The CNS dysfunction signs included listlessness, droopy ears, aimless walking, extreme excitability, foot stomping, goose-stepping, circling, a wide stance, laying down on one side, and leg paddling. Pony 1, which was inoculated with 4 logs of the virus, showed signs of anorexia on day 5 postinfection and signs of CNS dysfunction with anorexia and constipation by day 6 postinfection. The pony appeared to have irreversible CNS dysfunction signs on day 8 postinfection and was humanly killed. Pony 2, which was inoculated with 5 logs of virus, showed clinical signs of anorexia, scleritis, and ocular and nasal discharge on day 2 postinfection; improvement was seen on day 5 postinfection. The pony showed signs of CNS dysfunction on day 7 postinfection, but by day 9 postinfection it had recovered and appeared normal. Pony 3, which was inoculated with 6 logs of the virus, had nasal and ocular discharge on day 3 postinfection and anorexia and signs of CNS dysfunction at day 5 postinfection. This pony appeared to be in severe pain by day 8 postinfection and was humanely killed.

Lesions.

Macroscopic lesions.

No visible macroscopic lesions were observed in any parts of the viscera. Meninges of the brains of ponies 1 and 3 were slightly hyperemic.

Microscopic lesions.

Microscopic lesions were present in the brains of all three ponies. Ponies 1 and 3 had more extensive microscopic lesions associated with VEE IE virus infection than the pony 2, which survived. No lesions were observed in tissues of spinal cords of all three ponies. Mild to heavy monocytic (mostly lymphocytic) infiltrates were present in the meninges of ponies 1and 3, which were killed on day 8 postinfection (arrow in Figure 1A). However, mild meningitis was observed in pony 2, which survived the infection (Figure 1B, arrow). Mild to extensive perivascular cuffing with variable numbers of lymphocytes and macrophages (arrows in Figure 1C), heavy gliosis (arrow in Figure 1D), and perivascular cuffing of a small blood vessel (arrowhead in Figure 1D) were observed in the cerebrum, cerebellum, pons, thalamus, hippocampus, and medulla of ponies 1and 3. In pony 2, occasional mild to moderate perivascular cuffing of blood vessels was observed in the cerebrum, cerebellum, pons, and hippocampus (Figure 1E and arrow in Figure 1F) and gliosis was occasionally observed in the cerebrum, cerebellum, and pons. Neuronal degeneration (arrow in Figure 1G) and swollen glial cells (arrowhead in Figure 1G) were present in all brain tissues except the spinal cord in all three ponies. Occasional nonsuppurative focal necrosis was observed in cerebrum and pons of the ponies.

Virus isolation and identification.

Virus was isolated in Vero cell cultures from whole blood of ponies 1 and 2 on days 1–3 postinfection and from pony 3 only on days 1 and 2 postinfection. Virus was isolated from undiluted blood and blood diluted 1:10, but not from blood diluted 1:100 to 1: 10,000. However, virus was not isolated from brain homogenates and visceral organs of any of the ponies. In addition, sickness, paralysis, and death were not observed in any of the 3–5-day-old mice inoculated with brain samples. The virus isolates were confirmed as VEE IE by an IFA test with monoclonal antibodies (Table 1) and by a PRNT.

Serology.

Antibody (IgM) to homologous VEE virus antigen was first detected on day 5 postinfection in the serum of ponies 2 and 3 and on day 6 postinfection in the serum of pony 1 (Table 3). The IgM titer for homologous antigen increased rapidly in pony 1 (inoculated with 4 logs of virus) from 1:100 on day 6 postinfection to 1:10,000 on day 7 postinfection and remained at 1:10,000 on day 8 postinfection. The serum titer for VEE 1F viral antigen in this pony was 1:100 at day 6 postinfection and remained at this level until the pony was humanely killed at day 8 postinfection. The serum IgM titer of pony 1 for VEE IE Mena II viral antigen was 1:1,000 at day 7 postinfection and increased to 1:10,000 on day 8 postinfection. The serum IgM titers for VEE viral antigens ID and IAB in this pony were 1:1,000 at day 6 postinfection and increased to 1:10,000 on day 7 postinfection. Serum IgM antibody titers of 1:1,000 in pony 2 (inoculated with 5 logs of virus) were first detected at day 5 postinfection for homologous antigen NVSL93-42124 and ID and IAB antigens. Titers of 1:1,000 for Mena II and 1:100 for IF antigens were observed at day 6 postinfection. The titers increased to 1:10,000 the next day for NVSL 93-42124, ID, IAB, and Mena II antigens. However, they increased gradually for IF antigen, and on day 8 postinfection this titer increased to 1:10,000. In pony 3 (inoculated with 6 logs of virus), serum IgM titers of 1:1,000 to NVSL 93-42124, Mena II, and ID antigens and 1:100 for IAB antigen were detected at day 5 postinfection, which increased to 1:10,000 by day 7 postinfection. For VEE IF virus antigen, an IgM antibody titer of 1:100 was detected at day 6 postinfection, and this increased to 1:10,000 by day 8 postinfection.

Postinfection serum from the pony inoculated with TC-83 virus showed presence of IgM antibodies at day 8 postinfection to homologous antigen as well as to the heterologous antigens ID, IE NVSL 93-42124 and Mena II, but not for VEE virus IF antigen. However, titers for VEE ID and IE viral antigens were higher on day 8 and day 9 postinfection, respectively, than for homologous VEE TC-83 virus. The antibody titer increased gradually and by the day 13 postinfection, the IgM titers were 1:10,000 for TC-83, IE NVSL 93-42124, Mena II, and ID viral antigens and 1:1,000 for IF antigen.

Virus neutralizing antibody to homologous virus IE NVSL 93-42124 and IE human isolate Mena II was first detected on day 5 postinfection in the serum of pony 3 (Table 3). Ponies 1 and 2 showed virus neutralizing antibodies starting at day 6 postinfection. The antibody titers to both IE NVSL 93-42124 and Mena II viruses were similar (1:1,000 at day 6 postinfection and 1:10,000 at day 8 postinfection). Virus neutralizing antibodies to heterologous viruses IF and TC-83 were 10–100-fold less than the titers for homologous IE NVSL 93-42124 and Mena II viruses.

DISCUSSION

This study demonstrates the pathogenicity of VEE IE virus in ponies. The enzootic or endemic form of VEE IE virus had previously been considered avirulent for horses. The VEE IE virus NVSL 93-24124, which was isolated from the brain of a horse during an outbreak of VEE in Chiapas, Mexico, produced clinical disease in all three infected ponies, and the virus was isolated from their blood. However, this is not the first documented report of the pathogenicity of VEE virus serotype IE. Infections with VEE IE virus have been observed in horses, but were not recognized.15,16,24 The outbreak of VEE caused by this virus in horses in Tamaulipas and Veracruz, Mexico could not be established due to the lack of tissues for virus isolation.24 Subsequently, serologic studies showed that the outbreak was due to VEE serotype IE virus. In experimental studies,15,16 in spite of the clinical signs of infection with VEE virus, isolation of virus from serum and cerebrospinal fluid, and evidence of brain lesions in infected horses, the investigators concluded that the death of the horses was due to factors other than infection with VEE IE virus. Their conclusion was probably influenced by a previous concept that VEE serotype IE virus is nonpathogenic and that a viremia ≥ 105 suckling mouse intracerebral median lethal doses (SMICLD50) /ml in serum is necessary for the efficient transmission of VEE virus from infected horses to epizootic mosquitoes.2,36 The death of horses due to intrathecal inoculation of VEE IE virus 2177B was attributed to edema and anorexia, in spite of the fact that in the same study the signs and the gross and microscopic pathologic lesions were similar to those of horses inoculated with epizootic VEE IAB viruses. It is also possible that lack of knowledge of mutation(s) of viral genes, which can affect the virulence of the virus, might have contributed to their conclusion.37–41 In addition, the possibility of infection due to increased severity of tissue tropism cannot be ruled out.

The outbreak of VEE caused by the IE virus serotype in 1962 in Mexico coincided with an increase in the population of wild birds, rodents, pigeons, and mosquitoes.24 During the Chiapas outbreak of equine VEE (isolate VEE IE NVSL 93-24124) and the Oaxaca outbreak of VEE (isolate VEE IE NVSL 96-34286), an increase in the population of mosquitoes was observed. However, documentation of the increase in the population of rodents and birds and identification of the mosquito vectors and isolates of VEE virus are not available. It is possible that in the absence of an increase in the population of rodents and birds, which are reservoirs for VEE IE virus, a subtle change in the gene(s) of the virus in any carrier species, including disseminator mosquitoes, could have increased the virulence of enzootic VEE subtype IE virus for horses, resulting in the epidemic.26,42 Phylogenetic analysis showed that two epizootic VEE virus IE subtypes from Mexico were similar and related to enzootic viruses from Guatemala. It is also possible that a small, dormant subpopulation of epizootic virus is always present along with enzootic VEE IE virus, and under appropriate ecologic conditions, a new epizootic form of the virus may arise in the host, resulting in an epidemic in the horse population. Antibodies to VEE subtype IE virus were observed in the horse population of the endemic areas; thus, the possibility of horse-to-horse passage of an avirulent virus that becomes virulent cannot be ruled out.6

Two isolates of VEE subserotype IE virus from Chiapas (NVSL 93-42124) and Oaxaca (NVSL 96-32863), Mexico were isolated from brain samples of dead horses in 3–5-day-old suckling mice brains and also in a Vero cell line, but in the present study, virus was isolated in Vero cells from only blood of all three ponies The titer of the virus in the blood samples was low, since one pony inoculated with 6 logs of the virus showed a CPE with a 1:10 dilution of the blood only on the first day. No deaths were observed in five-day-old suckling mice inoculated intracerebrally with brain homogenates from the ponies. All three ponies had brain lesions, thus indicating viral infection. However, in a previous study,16 virus was not isolated from brain samples of a horse that died of infection with VEE subserotype IE virus (Nic2190) in suckling mice. In another study,15 virus was isolated from the brain tissues of horses that were inoculated with VEE IE virus (2177B) in suckling mice, but not from visceral tissues. Histopathologic lesions indicating encephalitis were found in the horses in both studies. The difference between these two studies was that in the study by Martin and others,16 the virus titers in the cerebrospinal fluid of the horses inoculated intrathecally with virus serotype 2177B were 2–2.4 logs higher than the virus titer in serum of the horse in the study by Dietz and others,15 which used virus serotype Nic 2190. In the present study, low amounts of virus were present in the blood samples of all three ponies, and the brains and visceral tissues did not have detectable levels of virus. Also, in human studies, virus was isolated only from serum of one patient who died of infection with enzootic VEE ID virus in 3–5-day-old mice, but inoculation of brain and viscera with VEE virus failed to yield any virus.9 In another human study, VEE ID virus was isolated from serum of two of eight patients in mice and in Vero cells.43 In contrast, infection with epizootic VEE viruses in horses produces a high concentration of virus in the serum of the animals, and viruses could be isolated from the brain tissues of most of the horses, as well as from visceral tissues.2,15,16

Microscopic pathology of various organs of horses that died of infection with the epizootic VEE IAB virus has been reported.6–8,10,11,15,16 However, the histopathology of only brain tissues from unrecognized VEE IE virus infections of horses that died of experimental virus infection were reported.15,16 In the present study with VEE IE 93-42124 virus, lesions that were observed in the CNS were similar to those observed in these two studies.15,16 The lesions were also similar to those observed in the CNS of the horses infected with epizootic VEE IAB virus, except for the absence of necrosis and infiltration of neutrophils. This could be due to the absence of a large amount of VEE IE virus in the brain that is needed to cause significant mortality of brain cells, which can lead to suppuration and infiltration of neutrophils. Also, in the present study, the two horses that had neurologic problems were humanely killed. It is possible that one or both of these horses could have recovered if they remained alive for a few more days. In any case, they had passed the acute phase of the infection when infiltration of the neutrophils would be expected. Histopathologic changes were not observed in any visceral tissues of the ponies, and VEE virus was not isolated from any visceral and brain tissues. This indicated that visceral tissues did not support virus multiplication to a level that can cause suppuration in the brain tissues and lesions in the visceral tissues. It is also possible that presence of only a minimal amount of virus in the brain can cause pathologic lesions or that VEE IE virus was neutralized by the immune response in the ponies.

The presence of IgM antibody to VEE virus was first detected at day 8 postinfection in the sera of a pony inoculated with VEE TC-83 virus and at days 5 and 6 postinfection in ponies inoculated with NVSL IE 93-42124 virus. This could be due to another factor because in 1993, sera of four VEE TC-83 IgM-positive horses on day 1 to day 11 postinfection (obtained from Dr. Jonathan F. Smith, U.S Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD) showed IgM antibodies to VEE virus at days 4–5 postinfection.

The IgM antibodies in sera of the ponies inoculated with NVSL VEE IE 93-42124 virus and with the TC-83 vaccine strain cross-reacted with the homologous and heterologous VEE virus antigens used in this study. In addition, sera obtained on days 8 and 9 postinfection from the pony inoculated with TC- 83 virus showed a higher IgM titer with heterologous VEE ID and IE viral antigens than with homologous TC-83 viral antigen. It is possible that IgM antibodies produced earlier reacted to a greater degree with common or cross-reacting epitopes on the E2 glycoproteins of subtype I VEE virus.30,31 Since the IgM titers of ponies for each VEE virus serosubtype (TC-83, ID, IE NVSL 93-42124, Mena II, and IF) were similar, it was impossible to distinguish between the homologous and heterologous viruses with this test.

The PRNT showed that homologous antiserum gave a fourfold or higher neutralizing antibody titer with homologous VEE virus than with heterologous VEE viruses.2 Similar results were also obtained in the present study. However, in cross-reaction studies with two different isolates of IE VEE viruses (68U201 and NVSL 93-42124), a four-fold or higher titer was obtained in sera from horses during an outbreak of VEE in Chiapas and Oaxaca, Mexico with homologous virus NVSL 93-42124 than with Guatemala viral isolate VEE IE 68U201.26 This indicated that virus IE 68U201 was serologically different from NVSL 93-42124 virus. In the present study, VEE IE virus 68U201 was not used, but no serologic difference was observed in virus neutralization titer to VEE IE NVSL 93-42124 and Mena II viruses. The explanation of these discrepancies awaits further studies. However, the possible existence of many variants of VEE IE virus subserotypes cannot be ruled out. Variants within VEE ID viruses have been previously reported.31

In the VEE virus complex, only two viruses belonging to serotype I, subtypes IAB and IC, are considered pathogenic to horses and humans. The horse is considered the primary and amplifying host of the virus, and humans are considered a dead end host.1,2 Recent studies on the pathogenicity of enzootic VEE viruses ID and IE and Tonate virus (subtype IIIB) suggest that all VEE viruses have the potential to cause infection and mortality in horses and humans.26,42–45 In the past, due to lack of sensitive laboratory techniques, many encephalitis cases were misdiagnosed as a non-VEE infection.15,16 However, current molecular techniques make possible the identification of a viral agent without isolation of the causative agent.26,43–45 Viral nucleic acid is synthesized early in the infection cycle before protein coats and before assembly of the virus. Use of the polymerase chain reaction (PCR) and nucleotide sequencing have made it possible to detect and identify viruses long before excretion of the infectious viral particles. In addition, the presence of viral nucleic acid in the blood and tissues of animals and invertebrates in the absence of signs and/or virus isolation have made identification of viruses possible by the application of extremely sensitive PCR or reverse transcriptase–PCR methods.26,43–49

The outbreak of VEE caused by subserotype IE virus NVSL 93-42124 in horses was observed in 1993 in the Pacific region of Chiapas, Mexico.26 Three years later, another outbreak of VEE in horses was observed in the adjoining northwestern state of Oaxaca, Mexico. The causative VEE IE virus (NVSL 96-32863) was antigenically indistinguishable with 1993 Chiapas VEE IE isolate (NVSL 93-24124). This indicated that the virus was moving northward and that there was a possibility of another outbreak in the near future in adjoining states. However, six years have passed and no outbreak of VEE has been reported. There may be several reasons for this. Mutation(s) in viral gene(s) causing reversion of the epizootic from to the enzootic form or low numbers of carrier rodents and birds and mosquito vectors can cause population changes in amplification and dissemination of VEE IE virus in horses. The presence of a small amount of epizootic virus variant along with the enzootic form of VEE ID viruses has been postulated.38 The VEE subserotype IC virus is known to remain dormant for many years before re-emerging and causing an epidemic.50 This may also be true for VEE virus subserotype IE isolate in Chiapas and Oaxaca. In endemic areas of Mexico and Central America, antibodies to VEE virus IE were observed. It is possible that high antibody titer for VEE IE virus in horses in the states adjoining Oaxaca, Mexico may afford protection from infection, thus diminishing the chance of a epidemic of VEE IE virus. Thus, tracking the movement of VEE IE virus becomes an important program for animal health and public health authorities in this region. Intensive case search programs, including animal and human hospitals, local clinics, physicians, and veterinarians of adjoining provinces of Oaxaca, are also necessary for early identification and control of infection with VEE virus subserotype IE.

In summary, VEE virus subserotype IE, which was considered to be avirulent, was found to be pathogenic for ponies. The VEE IE virus isolate NVSL 93-42124 from Oaxaca, Mexico, when inoculated into three ponies, caused clinical signs and histopathologic lesions that were observed only in brain tissues. In addition, VEE-specific immune responses were detected. The serum IgM antibodies detected had titers similar to those specific for VEE virus subserotypes TC-83 (IAB), IC, ID, Mena II, and IF and could not be used for the detection of VEE I viral serotypes. The virus neutralizing antibody titer was 10–100-fold greater for homologous virus compared with heterologous viruses. Virus neutralizing antibody titers to both VEE IE viruses NVSL 93-42124 and Mena II were similar. Thus, the mechanism for the neurovirulence of VEE IE viral strains is still not clear and requires further study.

Table 1

Identification of Venezuelan equine encephalomyelitis (VEE) subtype 1 viruses by an indirect fluorescent antibody (IFA) technique*

Monoclonal antibodies
VEE virus strainControlPolyclonal antibody1A3A-51A1B-93B4C-41A6C-3
* − = negative IFA result; + = positive IFA result. Polyclonal antibody to VEE virus was made in mouse ascitic fluid.
1AB++++
1C+++
1D+++
1E++
1F++
NVSL 93-42124++
NVSL 96-32863 (131,142)++
Table 2

Signs of Venezuelan equine encephalomyelitis (VEE) virus infection in ponies inoculated with VEE virus isolate IE NVSL 93-42124

VEE virus titer inoculatedDPI*Temperature (°C)Clinical signs
* DPI = days post infection.
† Killed on the day.
Pony 1 (4 logs)−137.1Normal
1–238.3Normal
3–438.6Normal
540Anorexia, depressed
6–739.8, 39.2Anorexia, constipated, goose-stepping, chewing, yawning, hypersensitive, weak
8†37.7Anorexia, staggering, falls and tries to get up
Pony 2 (5 logs)−1–037.7, 38.0Normal
139.5Normal
2–338.6, 39.3Nasal discharge, restless, scleritis, anorexia
4–538.3, 38.5Restless, normal appetite
6–839.5, 38.5Restless, aimless walking, foot stomping, droopy ears, normal appetite
9–11†37.7Normal
Pony 3 (6 logs)−1–037.6Normal
1–240.0, 38.1Normal
3–439.2, 40.3Nasal and ocular discharge, depressed
5–639.1, 38.3Anorexia, depressed, listlessness, droopy ears, aimless walking
738.3Very depressed, anorexia, aimless walking, extreme excitability, foot stomping, weak
8†38.1Staggering, falling down, lay down on one side and leg paddling
Table 3

Postinfection serologic reaction of ponies inoculated with Venezuelan equine encephalomyelitis (VEE) virus subserotype isolate IE NVSL 93-42124*

Serum IgM titer to VEE viral antigensSerum PRNT titer to VEE viral antigens
IEIABIEIAB
Ponies inoculated with VEE virus (type)DPI93-42124Mena-IIIDIFTC-8393-42124Mena-IIIDIFTC-83
* PRNT = plaque-reduction neutralization test; DPI = days postinfection; ND = not done.
Pony 1 (4 logs)0–4<100<100<100<100<100<10<10ND<10<10
5<100<100<100<100<100<10<10<10<10
6100<1001,0001001,0001,0001,0001010
710,0001,00010,00010010,0001,0001,000100100
810,00010,00010,00010010,00010,00010,000100100
Pony 2 (5 logs)0–4<100<100<100<100<100<10<10ND<10<10
51,000<1001,000<1001,000<10<10<10<10
610,0001,00010,00010010,0001,0001,0001010
710,00010,00010,0001,00010,0001,0001,000100100
810,00010,00010,00010,00010,00010,00010,0001,000100
910,00010,00010,00010,00010,00010,00010,0001,000100
Pony 3 (6 logs)0–4<100<100<100<100<100<10<10ND<10<10
51,0001,0001,000<1001001010<10<10
61,00010,00010,0001001,0001,0001,0001010
710,00010,00010,0001,00010,0001,0001,0001010
810,00010,00010,00010,00010,00010,00010,000100100
TC-83 virus (subtype IAB)
Pony 4 (7.5 logs)0–7<100<100<100<100<100<10<10ND<10<10
81001001,000<100100<10<10<1010
91,0001,0001,000<100100100101001,000
101,0001,00010,0001,00010,000100100101,000
1310,00010,00010,0001,00010,000100100101,000
Figure 1.
Figure 1. Figure 1.

Lesions in the cerebral cortexes of ponies inoculated with enzootic Venezuelan equine encephalomyelitis VEE IE NVSL 93-42124 virus isolate. A, Severe menengoencephalitis (arrow) in pony 3 (bar = 10 mm). B, Mild menengoencephalitis in pony 2, showing mostly infiltration of monocytes (arrow) (bar = 3.4 mm). C, Extensive perivascular cuffing in pony 1, showing infiltration of monocytes (arrows) (bar = 10 mm). D, Gliosis (arrow) and perivascular cuffing of a small blood vessel (arrowhead) in pony 1 (bar = 5 mm); E, Perivascular cuffing (rectangular area is shown in F) of a large blood vessel in pony 2 (bar = 10 mm). F, Enlargement of the rectangular area in E, showing mostly infiltration of lymphocytic cells (arrow) and endothelial cells lining of blood vessel (arrrowheads) (bar = 5 mm). G, Neuronal degeneration (arrows) and swollen glial cells (arrowheads) in pony 3 (bar = 5 mm). (Hematoxylin and eosin stained.)

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 68, 4; 10.4269/ajtmh.2003.68.485

Authors’ addresses: Sudhir P. Sahu, Douglas D. Pedersen, Allen L. Jenny, Beverly J. Schmitt, and Arnold D. Alstad, U.S. Department of Agriculture, Animal and Plant Health Inspection Service, National Veterinary Services Laboratories, Diagnostic Virology Laboratory, 1800 Dayton Avenue, Ames, IA 50010, Telephone: 515-663-7551, Fax: 515-663-7348, E-mail: sudhir.p.sahu@aphis.usda.gov

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