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The Hamster as an Animal Model for Eastern Equine Encephalitis— and Its Use in Studies of Virus Entrance into the Brain Slobodan Paessler,1,2 Patricia Aguilar,1,3 Michael Anishchenko,1,2 Hui-Qun Wang,4 Judith Aronson,1,2,3 Gerald Campbell,2 Ann-Sophie Cararra,1,2 and Scott C. Weaver1,2,3 Center for Biodefense and Emerging Infectious Diseases, and Departments of 2Pathology and 3Microbiology and Immunology, and 4Research Histology Core, National Institute of Environmental Health Services Center, University of Texas Medical Branch, Galveston
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Eastern equine encephalitis virus (EEEV) produces the most severe human arboviral diseases in the United States, with mortality rates of 30%–70%. Vasculitis associated with microhemorrhages in the brain dominates the pathological picture in fatal human eastern equine encephalitis, and neuronal cell death is detectable during the late stage of the disease. We describe use of the golden hamster to study EEEV-induced acute vasculitis and encephalitis. In hamsters, EEEV replicates in visceral organs, produces viremia, and penetrates the brain. The pathological manifestations and antigen distribution in the brain of a hamster are similar to those described in human cases of EEEV. Eastern equine encephalitis virus (EEEV) (family Togaviridae; genus Alphavirus) is an enveloped, positive-stranded RNA virus and causes the most severe human arboviral diseases in the United States, with high mortality rates [1, 2]. In North America, the virus circulates in swamps, among mosquitoes and passerine birds [3]. In human cases of encephalitis, fever, headache, vomiting, respiratory symptoms, leukocytosis, hematuria, seizures, and coma may occur [4]. Clinical studies of serologically confirmed eastern equine encephalitis in humans have shown, using magnetic resonance imaging (MRI) and computed tomography, changes in the basal ganglia and thalami that suggest brain edeReceived 24 April 2003; accepted 1 October 2003; electronically published 6 May 2004. Financial support: National Institutes of Health (NIH) (grant AI48807); NIH T32 Training Program in Emerging and Reemerging Infectious Diseases (AIO7536 to S.P.). Reprints or correspondence: Dr. Scott Weaver, Dept. of Pathology, Center for Tropical Diseases, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0605 (
[email protected]). The Journal of Infectious Diseases 2004; 189:2072–6 ! 2004 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2004/18911-0015$15.00
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ma, ischemia, and hypoperfusion during the early stage of disease [4]. Investigations of gross pathology in fatal human cases report brain edema with necrosis, facial or generalized edema, vascular congestion, and hemorrhage in the brain and visceral organs [1, 2, 5–9]. The predominant micropathological manifestations in the brain include vasculitis, hemorrhages, and encephalitis. The pathogenesis of EEEV is poorly understood, although data from experimental infection of mice [10], guinea pigs [11] and rhesus monkeys [12] and from histopathological studies of equine [13] and porcine [14] cases are available. Use of the mouse model for the study of alphavirus encephalitis is well established for several members of the genus [15], although this model generally lacks the ability to reproduce the vascular component of the disease [10]. In the present study, we report a hamster model of eastern equine encephalitis, with which to study acute vasculitis and encephalitis and to test antiviral drugs and vaccine candidates. Materials and methods. All work with live EEEV was conducted in biosafety level (BSL)–3 facilities, according to recommended procedures [16]; animal work was conducted by vaccinated persons. North American EEEV strain 79-2138 (1 passage in C6/36 cells, 1 passage in the brain of a suckling mouse), isolated from mosquitoes in 1979 in Massachusetts, was obtained from the World Reference Center for Arboviruses, at the University of Texas Medical Branch (Galveston). Female hamsters (Mesocricetus auratus) 6–8 weeks old were purchased from Harlan Sprague-Dawley. After 1 week of acclimatization in the BSL-3 facility, hamsters were inoculated subcutaneously, in the left medial thigh, with 103 pfu of EEEV in 0.1 mL of sterile PBS; the hamsters were then observed twice daily, for evidence of clinical illness. For virus titration in serum and organs, the hamsters were anesthetized with pentobarbital, and exsanguination was performed via cardiac puncture followed by perfusion with PBS; 2 control hamsters were inoculated with sterile PBS and were killed after 6 days. Each day, organs and blood from 2 hamsters were collected and homogenized in MEM containing 10% fetal bovine serum. A 10% suspension was maintained at !80"C until further processing. The amount of infectious virus was estimated by plaque assay [17]; 80% plaque-reduction neutralization tests were performed on Vero cells [17]. Tissue samples were fixed in 4% buffered formalin for 24 h and were stored in 70% ethanol for 12 h. The samples were then embedded in paraffin, were sectioned (into 5-mm-thick
slices), and were mounted on slides; standard hematoxylineosin (HE) staining was then performed. Sections were deparaffinized and rehydrated by use of xylene and graded-ethanol solutions. Slides were then treated with 3% hydrogen peroxide containing 0.05% sodium azide in PBS for 10 min, followed by microwave antigen retrieval at 100"C for 10 min, in Dako Target Retrieval Solution in an H2800 Microwave Processor (Energy Beam Sciences). After sequential 15-min incubations in 0.1% avidin and 0.01% biotin (Vector Laboratories), slides were incubated in 0.05% casein (Sigma)/0.05% Tween-20 (Dako)/PBS for 30 min, to block nonspecific protein binding. Murine hyperimmune serum (HIS) against EEEV was applied, at a 1:300 dilution, to sections for 60 min. To provide an antibody-negative control, the murine IgG-Ready-to-Use-Kit (InnoGenex) was used, at the same IgG concentration, on infected tissue; brain from uninfected hamsters was used as a negative control. The Histomouse-SP Kit (Zymed Laboratories) was used for detection of mouse antibody. Slides were counterstained with Mayer’s modified hematoxylin, before mounting and microscopy were performed. Results. After subcutaneous inoculation with 103 pfu of EEEV, all 10 hamsters developed fever at "24 h. Pressing of the head against the wall of the cage, vomiting, lethargy, and anorexia were observed 2–3 days after infection. All hamsters developed respiratory signs and more-progressive central nervous system (CNS) signs, including stupor and coma, on days 4–5 after infection; 6 hamsters died on day 6 after infection, and 4 animals died on day 5 after infection. EEEV was isolated from the brains of all hamsters; uninfected control hamsters did not show any detectable signs of disease. These results demonstrate that the EEEV used in the present study produced clinical encephalitis and fatal infection in all hamsters. In a parallel experiment, 2 EEEV-infected hamsters per day were killed, exsanguinated via cardiac puncture, and perfused with PBS. Visceral organs and brain were collected for virus titration. Beginning 24 h after infection, all hamsters showed the presence of infectious virus in serum for #3 days. Viremia peaked on day 2 after infection, and the lowest detectable level occurred on day 3 (figure 1A). On day 1 after infection, the largest amounts of virus were isolated from lung, liver, and muscle; on day 2, lung and liver showed an increase in titers of virus, and virus was also recovered from brain, heart, kidney, and spleen. On day 3, only titers of virus in heart and brain increased; however, from day 3 until death, the only organ with continuously increasing amounts of infectious virus was the brain (figure 1B). These results demonstrated the ability of EEEV to replicate in many different hamster organs, induce viremia, and penetrate the brain. Virus clearance from both the peripheral organs and serum correlated with the appearance of neutralizing antibody (figure 1A).
Daily examinations of gross pathology identified congestion in visceral organs (days 2–3), hepatomegaly and splenomegaly (days 2–3), hyperemia of the leptomeninges (days 3–6), and, during the late stage of disease, hemorrhages in the subarachnoid space (days 5–6). Pathological changes were not observed in uninfected control hamsters. Daily histological studies (2 hamsters/day) revealed hyperemia, vasculitis, and subependymal hemorrhages in the brain 24–48 h after infection. The localization of the early vascular manifestations (days 1–3) was periventricular, in the brain stem and basal ganglia. At this time, all neuronal cells appeared morphologically unchanged, and no inflammation in the brain parenchyma was detected. This vascular component in the brain was a dominant finding throughout the course of eastern equine encephalitis, becoming progressive with the appearance of neutralizing antibody (figure 2A). Infiltration of the bloodvessel walls by peripheral blood mononuclear cells and neutrophils associated with hemorrhages was observed. The inflammatory infiltrates were visible only during the late stage of infection and were formed mainly by macrophages, neutrophils, and microglia. Only a few lymphocytes, detected by HE staining, were present in the brain on days 5–6, and neuronal destruction associated with neuronophagia was visible on days 5–6. Vasculitis was also present in heart, liver, spleen, lung, kidney, and musculus quadriceps; vasculitis associated with microscopic hemorrhages in heart, liver, and lung of some of the hamsters became more prominent on days 4–6. Our histological results demonstrate an early onset of vasculitis in brain and in visceral organs after peripheral EEEV inoculation, indicating the importance of the vascular component of eastern equine encephalitis in the hamster model. Early involvement of the basal ganglia and brain stem was followed by late involvement of the cerebral cortex and cerebellum. Using murine EEEV HIS as a primary antibody, we detected viral antigen in the brains of all infected hamsters. No positive staining was observed in uninfected control hamsters. The first neuronal cells showing EEEV staining appeared on day 3 after infection, in the basal ganglia, brain stem, hippocampus, and midbrain; on days 4–6 most of the brain was involved (figure 2B). The strongest immunostaining during the end stage of disease was observed in the basal ganglia, midbrain, and brain stem. These results indicate rapid infection of neuronal cells in EEEV infection. Discussion. Apparent EEEV infection in humans often results in fatal encephalitis, with vascular, neuronal, and glial involvement. To our knowledge, no small-animal model of EEEV-induced vasculitis with vascular lesions and brain hemorrhages has been described [1, 2, 5–9]. Infection of mice produces encephalitis, but without the vascular manifestations typical of fatal human cases of the disease [10]. We have obtained similar results with National Institutes of Health Swiss mice BRIEF REPORT • JID 2004:189 (1 June) • 2073
Figure 1. A, Virus and antibody to eastern equine encephalitis virus (EEEV), in serum of hamsters after peripheral inoculation with EEEV. Titers of virus were measured, by plaque assay, in serum samples from 2 hamsters at each time point. After heat inactivation of the same serum samples, titers of antibody were estimated by plaque reduction–neutralization test (80% PRNT). B, Titers of virus in different organs excised from hamsters infected with EEEV. Titers were estimated, by plaque assay, at different time points after infection; at each time point, 2 hamsters were perfused with PBS, and organs were collected and homogenized. Final titers were calculated per gram of tissue. Vertical bars indicate SDs.
(data not shown), which develop encephalitis but fail to reproduce the acute, hemorrhagic vasculitis. In the present study, EEEV-infected hamsters developed viremia, followed by respiratory, gastrointestinal, and CNS signs and symptoms. Virus was isolated from many different organs, including the brain, and virus clearance from the peripheral blood correlated with the appearance of neutralizing antibody. However, the neutralizing antibody did not appear to be able to control virus replication in the brain. It is likely that this early antibody is IgM (not measured in the present study) and that the intracranially replicating virus is not accessible to this 2074 • JID 2004:189 (1 June) • BRIEF REPORT
antibody type. EEEV replication in the brains of hamsters was efficient and persisted throughout the infection. The onset of CNS disease correlated with the onset of vascular inflammation and microscopic hemorrhages in the basal ganglia, brain stem, and midbrain; these findings suggest that there is local hypoperfusion and hypoxia caused by vasculitis and the swelling of endothelial cells. It is interesting that previous studies also suggest that the early appearance of MRI-detectable human manifestations in the basal ganglia and brain stem is associated with hypoxia and inflammatory edema, rather than with virusmediated neuronal cell death [4]. However, we cannot exclude
Figure 2. A, Small hemorrhages in the brain stem of a hamster 5 days after inoculation with eastern equine encephalitis virus (EEEV). A highermagnification photomicrograph (inset) reveals the perivascular location of the hemorrhages and the absence of significant inflammation. B, Immunohistological section, stained with murine EEEV hyperimmune serum, of the cerebellum of a hamster, 5 days after inoculation with EEEV, showing infected Purkinje cells.
the possibility that our hamsters experienced virus-mediated neuronal dysfunction that went undetected. In hamster brain and visceral organs, the vascular component of eastern equine encephalitis was prominent; microhemorrhages were detected "48 h after infection. These findings are consistent with those from human cases of eastern equine encephalitis, where disruption of the blood-brain barrier is manifested by elevated protein concentrations and red blood cell counts in the cerebrospinal fluid [4]. The progression of vasculitis in hamster brain and visceral organs correlated with the appearance of neu-
tralizing antibody, and widely distributed microhemorrhages developed in several organs. This might indicate that immunemediated blood-vessel destruction plays a role in the later stages of EEEV infection. However, the early vascular manifestations and apparent disruption of vascular integrity that occur "1–3 days after infection are probably virus mediated and/or cytokine mediated, rather than antibody mediated. In our study, the neuronal phase of the disease was found to be rapid, and, prior to a hamster’s death, EEEV was found to establish productive infection in all parts of the brain. Our BRIEF REPORT • JID 2004:189 (1 June) • 2075
results suggest that early infection of periventricular and perivascular neuronal cells occurs in the basal ganglia and in the hippocampus. Venezuelan equine encephalitis virus penetrates the brain of infected mice via the olfactory nerve or other head nerves [18] and, during the early stage of disease, can be detected in the olfactory bulb [19]. In contrast, EEEV appears to rapidly invade the brain of infected hamsters via the blood, and the first antigen-positive neuronal cells are located in the basal ganglia and in the brain stem. The involvement of the olfactory bulb, during the end stage of hamster eastern equine encephalitis, suggests that this brain region is infected late and that the olfactory route is likely irrelevant to CNS penetration by EEEV. These findings are similar to those of studies of the early clinical manifestations of EEEV infections in humans [4]. Deresiewicz et al. suspected that the early radiologic changes represent either ischemia or edema, rather than tissue necrosis [4]. However, early pathological changes in hamsters, at the time of the onset of CNS disease, are characterized mainly by vasculitis associated with microhemorrhages. It is difficult to explain the early involvement of the basal ganglia and brain stem in hamsters; to our knowledge, no specific anatomic characteristics of these brain regions have been described that might explain their susceptibility to early infection [1, 2, 5–9]. In the present study, the inflammatory response in the brain was prominent in cases in which hamsters had survived for #5 days and appeared to be produced by macrophages and neutrophils. To identify these cells, specific staining for lymphocytes might be more sensitive than the method used in the present study. Unfortunately, because of the lack of immunological reagents for the hamster, these experiments could not be performed. The hamster model also displayed the peripheral pathological changes—including congestion and numerous microhemorrhages in the liver, spleen, and lung [1]—that have been described in fatal cases of human eastern equine encephalitis [1, 8, 20]. In summary, the hamster model appears to recapitulate the major findings (including vasculitis and encephalitis) of investigations of human cases of eastern equine encephalitis. Being an inexpensive animal model, it should be useful for the testing of antiviral drugs and vaccine candidates. Acknowledgments
We thank Robert Tesh, for providing the murine EEEV HIS and for his editorial help; Ilya Frolov, for scientific advice; and Ning-Ping Yang and Wenli Kang, for technical assistance.
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