Pathogenesis of experimental Ebola Zaire virus infection in BALB/c mice.

Guinea-pigs and non-human primates have traditionally been used as animal models for studying Ebola Zaire virus (EBO-Z) infections. The virus was also recently adapted to the stage of lethal virulence in BALB/c mice. This murine model is now in use for testing antiviral medications and vaccines. However, the pathological features of EBO-Z infection in mice have not yet been fully described. To identify sites of viral replication and characterize sequential morphological changes in BALB/c mice, adult female mice were infected with mouse-adapted EBO-Z and killed in groups each day for 5 days post-infection. Tissues were examined by light microscopy, immunohistochemistry, electron microscopy and in-situ hybridization. As in guinea-pigs and non-human primates, cells of the mononuclear phagocytic system were the earliest targets of infection. Viral replication was observed by day 2 in macrophages in lymph nodes and spleen. By the time of onset of illness and weight loss (day 3), the infection had spread to hepatocytes and adrenal cortical cells, and to macrophages and fibroblast-like cells in many organs. Severe lymphocytolysis was observed in the spleen, lymph nodes and thymus. There was minimal infection of endothelial cells. All of these changes resembled those observed in EBO-Z-infected guinea-pigs and non-human primates. In contrast to the other animal models, however, there was little fibrin deposition in the late stage of disease. The availability of immunodeficient, "gene-knockout" and transgenic mice will make the mouse model particularly useful for studying the early steps of Ebola pathogenesis.

Apoptosis induced in vitro and in vivo during infection by Ebola and Marburg viruses.

Induction of apoptosis has been documented during infection with a number of different viruses. In this study, we used transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling to investigate the effects of Ebola and Marburg viruses on apoptosis of different cell populations during in vitro and in vivo infections. Tissues from 18 filovirus-infected nonhuman primates killed in extremis were evaluated. Apoptotic lymphocytes were seen in all tissues examined. Filoviral replication occurred in cells of the mononuclear phagocyte system and other well-documented cellular targets by TEM and immunohistochemistry, but there was no evidence of replication in lymphocytes. With the exception of intracytoplasmic viral inclusions, filovirus-infected cells were morphologically normal or necrotic, but did not exhibit ultrastructural changes characteristic of apoptosis. In lymph nodes, filoviral antigen was co-localized with apoptotic lymphocytes. Examination of cell populations in lymph nodes showed increased numbers of macrophages and concomitant depletion of CD8+ T cells and plasma cells in filovirus-infected animals. This depletion was particularly striking in animals infected with the Zaire subtype of Ebola virus. In addition, apoptosis was demonstrated in vitro in lymphocytes of filovirus-infected human peripheral blood mononuclear cells by TEM. These findings suggest that lymphopenia and lymphoid depletion associated with filoviral infections result from lymphocyte apoptosis induced by a number of factors that may include release of various chemical mediators from filovirus-infected or activated cells, damage to the fibroblastic reticular cell conduit system, and possibly stimulation by a viral protein.

Pathogenesis of experimental Ebola virus infection in guinea pigs.

The subtype Zaire of Ebola (EBO) virus (Mayinga strain) was adapted to produce lethal infections in guinea pigs. In many ways, the disease was similar to EBO infections in nonhuman primates and humans. The guinea pig model was used to investigate the pathologic events in EBO infection that lead to death. Analytical methods included immunohistochemistry, in situ hybridization, and electron microscopy. Cells of the mononuclear phagocyte system, primarily macrophages, were identified as the early and sustained targets of EBO virus. During later stages of infection, interstitial fibroblasts in various tissues were infected, and there was evidence of endothelial cell infection and fibrin deposition. The distribution of lesions, hematologic profiles, and increases in serum biochemical enzymes associated with EBO virus infection in guinea pigs was similar to reported findings in experimentally infected nonhuman primates and naturally infected humans.

Evaluation of immune globulin and recombinant interferon-alpha2b for treatment of experimental Ebola virus infections.

A passive immunization strategy for treating Ebola virus infections was evaluated using BALB/ c mice, strain 13 guinea pigs, and cynomolgus monkeys. Guinea pigs were completely protected by injection of hyperimmune equine IgG when treatment was initiated early but not after viremia had developed. In contrast, mice were incompletely protected even when treatment was initiated on day 0, the day of virus inoculation. In monkeys treated with one dose of IgG on day 0, onset of illness and viremia was delayed, but all treated animals died. A second dose of IgG on day 5 had no additional beneficial effect. Pretreatment of monkeys delayed onset of viremia and delayed death several additional days. Interferon-alpha2b (2 x 10(7) IU/kg/day) had a similar effect in monkeys, delaying viremia and death by only several days. Effective treatment of Ebola infections may require a combination of drugs that inhibit viral replication in monocyte/macrophage-like cells while reversing the pathologic effects (e.g., coagulopathy) consequent to this replication.

Marburg hemorrhagic fever: report of a case studied by immunohistochemistry and electron microscopy.

The histologic and ultrastructural findings in a fatal human case of Marburg hemorrhagic fever are reported. Marburg virus was isolated from fluids and tissues and was identified in tissues by immunohistochemistry and electron and immunoelectron microscopy. The distribution of viral antigen by light level immunohistochemistry correlated with histologic lesions and also with the ultrastructural localization of virions. The tissue distribution and lesions of Marburg virus in this patient were consistent with the disease described in other human Marburg infections. Immunocytochemistry and ultrastructural examination revealed several previously unreported findings. A striking predilection for viral infection of the pancreatic islet cells was noted. In other tissues, macrophages were the primary cellular target for Marburg virus infection, with hepatocytes, adrenal cortical and medullary cells, and fibroblast-like cells also serving as important sites of viral replication. This case demonstrates the value of transmission electron microscopy as a tool for assisting in the definitive diagnosis of Marburg or Ebola hemorrhagic fever, as well as providing insight into the pathogenesis of these agents.

Pathology of experimental Ebola virus infection in African green monkeys. Involvement of fibroblastic reticular cells.

BACKGROUND: Ebola virus has been responsible for explosive lethal outbreaks of hemorrhagic fever in both humans and nonhuman primates. Previous studies showed a predilection of Ebola virus for cells of the mononuclear phagocyte system and endothelial cells. OBJECTIVE: To examine the distribution of lesions and Ebola virus antigen in the tissues of six adult male African green monkeys (Cercopithecus aethiops) that died 6 to 7 days after intraperitoneal inoculation of Ebola-Zaire (Mayinga) virus. METHODS: Tissues were examined histologically, immunohistochemically, and ultrastructurally. RESULTS: A major novel finding of this study was that fibroblastic reticular cells were immunohistochemically and ultrastructurally identified as targets of Ebola virus infection. CONCLUSIONS: The role of Ebola virus-infected fibroblastic reticular cells in the pathogenesis of Ebola hemorrhagic fever warrants further investigation. This is especially important because of recent observations indicating that fibroblastic reticular cells, along with the reticular fibers they produce, maximize the efficiency of the immune response.

Lethal experimental infection of rhesus monkeys with Ebola-Zaire (Mayinga) virus by the oral and conjunctival route of exposure.

OBJECTIVE: The source of infection or mode of transmission of Ebola virus to human index cases of Ebola fever has not been established. Field observations in outbreaks of Ebola fever indicate that secondary transmission of Ebola virus is linked to improper needle hygiene, direct contact with infected tissue or fluid samples, and close contact with infected patients. While it is presumed that the virus infects through either breaks in the skin or contact with mucous membranes, the only two routes of exposure that have been experimentally validated are parenteral inoculation and aerosol inhalation. Epidemiologic evidence suggests that aerosol exposure is not an important means of virus transmission in natural outbreaks of human Ebola fever; this study was designed to verify that Ebola virus could be effectively transmitted by oral or conjunctival exposure in nonhuman primates. MATERIALS AND METHODS: Adult rhesus monkeys (Macaca mulatta) were exposed to Ebola-Zaire (Mayinga) virus orally (N=4), conjunctivally (N=4), or by intramuscular inoculation (N=1, virus-positive control). RESULTS: Four of four monkeys exposed by the conjunctival route, three of four monkeys exposed by the oral route, and the intramuscularly inoculated positive control monkey (one of one) were successfully infected with Ebola-Zaire (Mayinga). Seven monkeys died of Ebola fever between days 7 and 8 postexposure. One monkey was given aggressive supportive therapy and a platelet transfusion; it lived until day 12 postexposure. CONCLUSIONS: Findings in this study experimentally confirm that Ebola virus can be effectively transmitted via the oral or conjunctival route of exposure in nonhuman primates.

Experimental infection of cynomolgus macaques with Ebola-Reston filoviruses from the 1989-1990 U.S. epizootic.

This study describes the pathogenesis of the Ebola-Reston (EBO-R) subtype of Ebola virus for experimentally infected cynomolgus monkeys. The disease course of EBO-R in macaques was very similar to human disease and to experimental diseases in macaques following EBO-Zaire and EBO-Sudan infections. Cynomolgus monkeys infected with EBO-R in this experiment developed anorexia, occasional nasal discharge, and splenomegaly, petechial facial hemorrhages and severe subcutaneous hemorrhages in venipuncture sites, similar to human Ebola fever. Five of the six EBO-R infected monkeys died, 8 to 14 days after inoculation. One survived and developed high titered neutralizing antibodies specific for EBO-R. The five acutely ill monkeys shed infectious virus in various bodily secretions. Further, abundant virus was visualized in alveolar interstitial cells and free in the alveoli suggesting the potential for generating infectious aerosols. Thus, taking precautions against aerosol exposures to filovirus infected primates, including humans, seems prudent. This experiment demonstrated that EBO-R was lethal for macaques and was capable of initiating and sustaining the monkey epizootic. Further investigation of this animal model should facilitate development of effective immunization, treatment, and control strategies for Ebola hemorrhagic fever.

Pathology of experimental inhalation anthrax in the rhesus monkey.

BACKGROUND: The inhalation form of anthrax, although rare, is nearly always fatal because of the rapid progression of the disease with little host response until the terminal stages of the disease. The Gulf War heightened the concern that anthrax could be used as a biologic weapon. Past studies modeling pathologic changes in human inhalation anthrax have used the rhesus monkey. EXPERIMENTAL DESIGN: We studied pathologic changes in the rhesus monkey model of inhalation anthrax. Gross examination as well as light and electron microscopy were used to define pathologic alterations. Immunolabeling techniques were used to identify the anthrax bacillus by light and electron microscopy. RESULTS: Gross changes included hemorrhage in mesenteric (54%) and tracheobronchial (46%) lymph nodes, meninges (38%), lungs (31%), and small intestinal serosa (31%). Histopathologic changes included suppurative meningitis (77%); hemorrhages in the meninges (54%), neuropil (31%), and pulmonary alveoli (31%); and pneumonia (15%). Spleens and various lymph nodes from all monkeys had one or more of the following changes: hemorrhage, acute inflammation, extracellular bacilli, lymphocytic depletion, and histiocytosis. Spleens of two monkeys were devoid of extracellular bacilli, but degraded intrahistiocytic bacilli reacted with Ab to Bacillus anthracis cell wall polysaccharide. CONCLUSIONS: In our study, compared with previous reports, meningitis and mesenteric lymph node hemorrhages were more common, whereas mediastinal and tracheobronchial lymph node hemorrhages were less common. Immunostaining highlighted intracellular bacilli that would have been otherwise missed by light microscopic examination.

Dizocilpine (MK-801) arrests status epilepticus and prevents brain damage induced by soman.

The involvement of the NMDA receptor in the neurotoxicity induced by soman, an organophosphorus compound which irreversibly inhibits cholinesterase, was studied in guinea pigs. The drug MK-801 (0.5, 1 or 5 mg/kg, i.p.) was given as a pretreatment before a convulsant dose of soman or as a posttreatment (30, 100 or 300 micrograms/kg, i.m.) 5 min after the development of soman-induced status epilepticus. Pyridostigmine, atropine and pralidoxime chloride were also given to each subject to counteract the lethality of soman. All subjects that were challenged with soman and given the vehicle for MK-801 (saline) exhibited severe convulsions and electrographic seizure activity. Neuronal necrosis was found in the hippocampus, amygdala, thalamus and the pyriform and cerebral cortices of those subjects surviving for 48 hr. Pretreatment with 0.5 or 1 mg/kg doses of MK-801 did not prevent nor delay the onset of seizure activity but did diminish its intensity and led to its early arrest. At the largest dose (5 mg/kg), MK-801 completely prevented the development of seizure activity and brain damage. Posttreatment with MK-801 prevented, arrested or reduced seizure activity, convulsions and neuronal necrosis in a dose-dependent manner. The NMDA receptor may play a more critical role in the spread and maintenance, rather than the initiation of cholinergically-induced seizure activity.

Decreased brain pathology in organophosphate-exposed rhesus monkeys following benzodiazepine therapy.

Two benzodiazepine compounds, midazolam and diazepam, were administered as adjunctive treatment to soman-exposed rhesus monkeys to evaluate their effects on acute soman intoxication. Monkeys were pretreated orally with pyridostigmine, exposed to soman, and treated i.m. with atropine, pralidoxime chloride (2-PAM), and with midazolam, diazepam or sterile water (control). All monkeys that received the benzodiazepines recovered sooner and exhibited no convulsions. Neuronal degenerative and necrotic lesions were decreased or eliminated in the entorhinal cortex, caudate nucleus, and hippocampus of those animals that received benzodiazepine therapy. These findings support the continued evaluation of drugs with anticonvulsant activity as standard adjunct therapy for soman intoxication.

Coronavirus-like virions associated with a wasting syndrome in guinea pigs.

An apparent wasting syndrome was observed in newly arriving 3 to 4 week old guinea pigs characterized by anorexia, weight loss, diarrhea, perineal staining and death. Diagnostic efforts to attribute the disease to husbandry, environmental factors or to known guinea pig pathogens were unsuccessful. Clinical signs, enteric histopathological lesions and diagnostic transmission electron microscopy identification of typical coronavirus-like virions in fecal samples were consistent with enteric coronaviral diseases seen in other species.

Atropine and/or diazepam therapy protects against soman-induced neural and cardiac pathology.

Toxic doses of the organophosphonate anticholinesterase agent soman can produce neural and cardiac lesions in animals that survive the acute poisoning. The ability of two standard antidote drugs, atropine and diazepam, along with the oxime pralidoxime (2-PAM) Cl, were evaluated for their ability to block these pathological effects. Rats were challenged with a fixed dose (85 micrograms/kg, sc) of soman and treated im 5 min later with 25 mg/kg 2-PAM Cl and one of the following combinations of atropine (0.0, 1.0, 3.2, 10.0, or 32.0 mg/kg) and diazepam (0.0, 0.1, 0.32, 1.0, or 3.2 mg/kg) in a balanced design. The severity of acute anticholinesterase intoxication signs was rated 1 hr after exposure; Body weights and behavioral reactivity ratings were obtained daily for 16 days after exposure; brains and hearts of all surviving subjects were then evaluated for pathological changes. Soman challenge resulted in 33% lethality in animals that received only 2-PAM therapy; both atropine and diazepam reduced lethality in a dose-dependent fashion. Across all treatment conditions greater than 50% of the deaths occurred later than 24 hr after intoxication and treatment. Acute intoxication signs were differentially moderated by the two drugs: atropine reduced all six signs in a dose-dependent fashion; diazepam had no effect on lacrimation and eye bulb protrusion, antagonized signs of salivation and motor abnormalities in a dose-dependent manner, and antagonized the effects of soman on signs of physical activity and coordination only at low doses. All doses of diazepam and the highest dose of atropine moderated body weight loss and a syndrome of behavioral hyperreactivity observed after exposure. Brain pathology was significantly reduced by all doses of diazepam and/or the highest dose of atropine, but no single drug or drug combination was effective in protecting all animals in a group from some brain pathology. Both drugs blocked the development of cardiac lesions in a dose-dependent fashion. The results demonstrate that diazepam or high doses of atropine can antagonize the development of brain lesions that result from soman exposure. Pharmacological management of epileptiform motor abnormalities during the acute intoxication is critical for this effect. In contrast, soman-induced cardiac pathology may occur secondarily as a consequence of the severe brain lesions or develop independently of brain lesion formation due possibly to sympathetic overstimulation.

Effect of soman exposure on the acquisition of an operant alternation task.

Following recovery from soman administration, rats were trained on an operant alternation task with a time-out between response periods. As animals became proficient at the task, both the operant requirements and the length of time-out periods were gradually raised to Fixed Ratio 20 with a 20-second Intertrial Interval. Training sessions continued until animals attained criterion or 100 training sessions had been given. Soman produced a dose-related lethality and signs of cholinergic hyperstimulation. Although all saline controls and 90% of animals receiving 75 micrograms/kg soman attained terminal performance, only one-third of the animals given either 85 or 95 micrograms/kg soman were able to learn this task. Sessions to attain criterion performance produced similar dose-dependent results: All saline animals attained criterion, while only 60%, 33% and 33% of the animals given 75, 85 or 95 micrograms/kg soman respectively reached criterion. Additionally, both 85 and 95 micrograms/kg soman produced severe neural lesions, including cortical atrophy and ventricular dilation.

Cardiomyopathy in Soman and Sarin intoxicated rats.

Rats surviving various single dose of the organophosphorus anticholinesterase nerve agents Soman and Sarin were examined by light microscopy at intervals up to 35 days post-exposure. Brain lesions, identical to those that have been reported elsewhere were present, as well as a previously unreported finding associated with Soman or Sarin intoxication: half of all animals that had brain lesions also had areas of myocardial degeneration and necrosis. Depending upon the point in time at which cardiac tissues were examined, findings varied from areas of acute myolysis and necrosis to areas undergoing resolution of damage. The finding of brain lesions in those animals having cardiac lesions suggests a relationship between the convulsion induced neurologic and cardiac lesions. These studies suggest that convulsive doses of chemical warfare agents induce pathological changes in the cardiovascular system of laboratory animals.

Effect of prior influenza virus infections on susceptibility of AKR/J mice to respiratory challenge with Legionella pneumophila.

Influenza virus administered intranasally to AKR/J mice, followed 3 days later by Legionella pneumophila inoculated intranasally, caused significantly greater mortality than did either of the two agents administered alone. Viable concentrations of both bacteria and viruses dropped in sequentially infected animals, despite the ultimate fatal outcome. Viral concentrations, however, did not decrease as rapidly in sequentially infected as in singly infected mice. Histopathologic lesions were consistent with viral replication aided by elaboration of a bacterial toxin. This observation contrasts with the more commonly observed sequence in which the bacterium proliferates after the virus interferes with host defense. Cell-free preparations were found to have toxic activity.