The pathology of experimental aerosolized monkeypox virus infection in cynomolgus monkeys (Macaca fascicularis).

Cynomolgus monkeys (Macaca fascicularis) were exposed by fine-particle aerosol to lethal doses of monkeypox virus, Zaire strain. Death, attributable to fibrinonecrotic bronchopneumonia, occurred 9 to 17 days postexposure. Lower airway epithelium served as the principal target for primary infection. The relative degree of involvement among lymphoid tissues suggested that tonsil, mediastinal, and mandibular lymph nodes were also infected early in the course of the disease, and may have served as additional, although subordinate, sites of primary replication. The distribution of lesions was consistent with lymphatogenous spread to the mediastinal lymph nodes and systemic dissemination of the virus through a monocytic cell-associated viremia. This resulted in lesions affecting other lymph nodes, the thymus, spleen, skin, oral mucosa, gastrointestinal tract, and reproductive system. The mononuclear phagocyte/dendritic cell system was the principal target within lymphoid tissues and may also have provided the means of entry into other systemic sites. Hepatic involvement was uncommon. Lesions at all affected sites were characterized morphologically as necrotizing. Terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining of select lesions suggested that cell death within lymphoid and epithelial tissues was due in large part to apoptosis. Skin and mucosal surfaces of the respiratory and gastrointestinal tracts also exhibited variable proliferation of epithelial cells and subjacent fibroblasts. Epithelial intracytoplasmic inclusion bodies, consistent with Guarnieri bodies, were usually inconspicuous by light microscopy, but when present, were most readily apparent in the stratified squamous epithelium of the oral mucosa and epidermis. Multinucleated syncytial cells were also occasionally observed in the stratified squamous epithelium of the tongue, tonsil, and skin, and in the intestinal mucosa. Monkeypox virus antigen was readily demonstrated by immunohistochemistry using anti-vaccinia mouse polyclonal antibodies as well as anti-monkeypox rabbit polyclonal antibodies. Detectable poxviral antigen was limited to sites exhibiting obvious morphologic involvement and was most prominent within epithelial cells, macrophages, dendritic cells, and fibroblasts of affected tissues. The presence of poxviral antigen, as determined by immunohistochemistry, correlated with ultrastructural identification of replicating virus. Concurrent bacterial septicemia, present in one monkey, was associated with increased dissemination of the virus to the liver, spleen, and bone marrow and resulted in a more rapidly fatal clinical course.

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.

Pathology of fatal West Nile virus infections in native and exotic birds during the 1999 outbreak in New York City, New York.

West Nile fever caused fatal disease in humans, horses, and birds in the northeastern United States during 1999. We studied birds from two wildlife facilities in New York City, New York, that died or were euthanatized and were suspected to have West Nile virus infections. Using standard histologic and ultrastructural methods, virus isolation, immunohistochemistry, in situ hybridization and reverse-transcriptase polymerase chain reaction, we identified West Nile virus as the cause of clinical disease, severe pathologic changes, and death in 27 birds representing eight orders and 14 species. Virus was detected in 23/26 brains (88%), 24/ 25 hearts (96%), 15/18 spleens (83%), 14/20 livers (70%), 20/20 kidneys (100%), 10/13 adrenals (77%), 13/ 14 intestines (93%), 10/12 pancreata (83%), 5/12 lungs (42%), and 4/8 ovaries (50%) by one or more methods. Cellular targets included neurons and glial cells in the brain, spinal cord, and peripheral ganglia; myocardial fibers; macrophages and blood monocytes; renal tubular epithelium; adrenal cortical cells; pancreatic acinar cells and islet cells; intestinal crypt epithelium; oocytes; and fibroblasts and smooth muscle cells. Purkinje cells were especially targeted, except in crows and magpies. Gross hemorrhage of the brain, splenomegaly, meningoencephalitis, and myocarditis were the most prominent lesions. Immunohistochemistry was an efficient and reliable method for identifying infected cases, but the polyclonal antibody cross-reacted with St. Louis encephalitis virus and other flaviviruses. In contrast, the in situ hybridization probe pWNV-E (WN-USAMRIID99) reacted only with West Nile virus. These methods should aid diagnosticians faced with the emergence of West Nile virus in the United States.

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.

Ebola (subtype Reston) virus among quarantined nonhuman primates recently imported from the Philippines to the United States.

In April 1996, laboratory testing of imported nonhuman primates (as mandated by quarantine regulations) identified 2 cynomolgus macaques (Macaca fascicularis) infected with Ebola (subtype Reston) virus in a US-registered quarantine facility. The animals were part of a shipment of 100 nonhuman primates recently imported from the Philippines. Two additional infected animals, who were thought to be in the incubation phase, were identified among the remaining 48 animals in the affected quarantine room. The other 50 macaques, who had been held in a separate isolation room, remained asymptomatic, and none of these animals seroconverted during an extended quarantine period. Due to the rigorous routine safety precautions, the facility personnel had no unprotected exposures and remained asymptomatic, and no one seroconverted. The mandatory quarantine and laboratory testing requirements, put in place after the original Reston outbreak in 1989-1990, were effective for detecting and containing Ebola virus infection in newly imported nonhuman primates and minimizing potential human transmission.

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.

Pulmonary lesions in experimental ophidian paramyxovirus pneumonia of Aruba Island rattlesnakes, Crotalus unicolor.

Histologic and ultrastructural changes were observed in the respiratory portions of lung in five 29-40-month-old Aruba Island rattlesnakes, Crotalus unicolor, that were inoculated with an Aruba Island Rattlesnake virus (AIV) strain of ophidian paramyxovirus (OPMV) isolated from an Aruba Island rattlesnake. Lungs from one non-infected and three mock-infected Aruba Island rattlesnakes were examined also. From 4 to 22 days following intratracheal inoculation, progressive microscopic changes were seen in the lung. Initially, increased numbers of heterophils were observed in the interstitium followed by proliferation and vacuolation of epithelial cells lining faveoli. The changes appeared to progress from cranial to caudal portions of the respiratory lung following inoculation. Beginning at 4 days postinoculation, viral antigen was demonstrated in epithelial cells lining faveoli with an immunofluorescent technique using a rabbit anti-AIV polyclonal antibody. Electron microscopy revealed loss of type I cells, hyperplasia of type II cells, and interstitial infiltrates of heterophils and mononuclear cells. Viral nucleocapsid material was seen within the cytoplasm and mature virus was seen budding from cytoplasmic membranes of infected type I and type II cells from 8 to 19 days after infection. A virus consistent with AIV was isolated from lung tissues of infected rattlesnakes, thus fulfilling Koch's postulates.

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.

Experimental infection of guinea pigs with Venezuelan hemorrhagic fever virus (Guanarito): a model of human disease.

Venezuelan hemorrhagic fever (VHF), a newly described disease caused by an arenavirus (Guanarito), has resulted in multiple human deaths in Venezuela. To develop an animal model of this disease, strain 13 and Hartley strain guinea pigs were inoculated subcutaneously with Guananto strain 95551 of arenavirus in a pilot study to determine susceptibility of the species to the virus. All animals were killed when moribund 12-14 days following inoculation. Animals were necropsied and tissues were fixed and examined by both light and electron microscopy. Viral antigen was demonstrated in the tissues by immunohistochemistry at both the light and electron microscopic levels. Lesions were characterized by single cell necrosis of epithelium of the gastrointestinal tract, interstitial pneumonia, lymphoid and hematopoietic cell necrosis, and the presence of platelet thrombi in occasional blood vessels associated with hemorrhage. Viral antigen was demonstrated in lymphoid tissues and macrophages, endothelial cells of multiple organs, pulmonary epithelium, epithelium of the gastrointestinal tract, and in miscellaneous other tissues and cells. Intact virions and typical arenavirus inclusions were demonstrated by immunoelectron microscopy in these tissues. Based on these findings, the guinea pig appears to be a valid animal model of the human disease.

Characterization of Oliveros virus, a new member of the Tacaribe complex (Arenaviridae: Arenavirus).

Oliveros virus is an agent isolated in cell culture from Bolomys obscurus (Rodentia, Muridae, Sigmodontinae) captured on the central Argentine pampa. Oliveros virus was shown to be related to members of the Tacaribe complex of the family Arenaviridae by immunofluorescent antibody (IFA) tests, electrophoretic pattern of viral proteins, and morphology as observed by electron microscopy. It was distinct from 12 other arenaviruses by a combination of plaque-reduction neutralization tests, comparison of endpoint titers among cross-IFA tests, and comparison of viral RNA sequence data. This agent is the third new arenavirus from South America described within the last three years.

Characterization of a new Marburg virus isolated from a 1987 fatal case in Kenya.

In 1987, an isolated case of fatal Marburg disease was recognized during routine clinical haemorrhagic fever virus surveillance conducted in Kenya. This report describes the isolation and partial characterization of the new Marburg virus (strain Ravn) isolated from this case. The Ravn isolate was indistinguishable from reference Marburg virus strains by cross-neutralization testing. Virus particles and aggregates of Marburg nucleocapsid matrix in Ravn-infected vero cells, were visualized by immunoelectron microscopic techniques, and also in tissues obtained from the patient and from inoculated monkeys. The cell culture isolate produced a haemorrhagic disease typical of Marburg virus infection when inoculated into rhesus monkeys. Disease was characterized by the sudden appearance of fever and anorexia within 4 to 7 days, and death by day 11. Comparison of nucleotide sequences for portions of the glycoprotein genes of Marburg-Ravn were compared with Marburg reference strains Musoki (MUS) and Popp (POP). Nucleotide identity in this alignment between RAV and MUS is 72.3%, RAV and POP is 71%, and MUS and POP is 91.7%. Amino acid identity between RAV and MUS is 72%, RAV and POP is 67%, and MUS and POP is 93%. These data suggest that Ravn is another subtype of Marburg virus, analogous to the emerging picture of a spectrum of Ebola geographic isolates and subtypes.

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.

Differentiation of filoviruses by electron microscopy.

Cultured monolayers of MA-104, Vero 76, SW-13, and DBS-FRhL-2 cells were infected with Marburg (MBG), Ebola-Sudan (EBO-S), Ebola-Zaire (EBO-Z), and Ebola-Reston (EBO-R) viruses (Filoviridae, Filovirus) and examined by electron microscopy to provide ultrastructural details of morphology and morphogenesis of these potential human pathogens. Replication of each filovirus was seen in all cell systems employed. Filoviral particles appeared to enter host cells by endocytosis. Filoviruses showed a similar progression of morphogenic events, from the appearance of nascent intracytoplasmic viral inclusions to formation of mature virions budded through plasma membranes, regardless of serotype or host cell. However, ultrastructural differences were demonstrated between MBG and other filoviruses. MBG virions recovered from culture fluids were uniformly shorter in mean unit length than EBO-S, EBO-Z, or EBO-R particles. Examination of filovirus-infected cells revealed that intermediate MBG inclusions were morphologically distinct from EBO-S, EBO-Z, and EBO-R inclusions. No structural difference of viral inclusion material was observed among EBO-S, EBO-Z, and EBO-R. Immunoelectron microscopy showed that the filoviral matrix protein (VP40) and nucleoprotein (NP) accumulated in EBO-Z inclusions, and were closely associated during viral morphogenesis. These details facilitate the efficient and definitive diagnosis of filoviral infections by electron microscopy.

Use of immunoelectron microscopy to demonstrate Francisella tularensis.

Three immunoelectron microscopy (IEM) methods were employed to show laboratory-cultivated Francisella tularensis. By the IEM assays, F. tularensis was distinguished from four antigenically distinct gram-negative bacteria. IEM should be a valuable tool for confirming presumptive isolates of F. tularensis and may potentially be useful for demonstrating other medically important bacteria.

Association of Ebola-related Reston virus particles and antigen with tissue lesions of monkeys imported to the United States.

During 1989-1990, an epizootic involving a filovirus closely related to Ebola virus occurred in a Reston, Virginia, primate-holding facility. Tissues were collected from cynomolgus monkeys and examined by electron microscopy and immunohistochemistry for Ebola-related viral antigen. Viral replication was extensive in fixed tissue macrophages, interstitial fibroblasts of many organs, circulating macrophages and monocytes, and was observed less frequently in vascular endothelial cells, hepatocytes, adrenal cortical cells and renal tubular epithelium. Viral replication was observed infrequently in epithelial cells lining ducts or mucous membranes, intestinal epithelial cells, eosinophils and plasma cells. Replication of Reston virus in lymphocytes was never observed, in contrast to reports of lymphocytes of monkeys experimentally infected with the Ebola-Zaire virus. Free filoviral particles were seen in pulmonary alveoli and renal tubular lumina, which correlates with epidemiological evidence of droplet and fomite transmission. Viral infection of interstitial fibroblasts and macrophages caused multisystemic disruptive lesions involving connective tissue. Focal necrosis in organs where viral replication was minimal may have been secondary to ischaemia caused by fibrin deposition and occasional platelet-fibrin thrombi. Immunoelectron microscopy on sections of liver, differentiated viral tubular inclusion masses and precursor material from non-viral tubuloreticular inclusions. Immunohistochemistry showed that the distribution of viral antigen in affected tissue correlated well with ultrastructural localization of virions.

Rapid identification of Ebola virus and related filoviruses in fluid specimens using indirect immunoelectron microscopy.

Recent filoviral outbreaks in animal primates have raised public awareness of the potential for filoviruses to become a public health concern; methods that efficiently identify these viruses are therefore of high priority. An indirect immunoelectron microscopy method, which uses homologous guinea pig polyclonal antiserum, successfully identified Ebola-related (Reston) virus particles in serum and tissue culture fluid specimens with infectivity titres of 300 plaque forming units (pfu) per ml or more. The sensitivity of this procedure is sufficient to show virus in most acute phase sera, and is equal to that of the antigen capture enzyme linked immunosorbent assay (ELISA). The immunoelectron microscopy fluid technique can differentiate among antigenically distinct filoviruses in less than three hours. It should be valuable in the rapid diagnosis of potential filoviral infections.

Toxicity and kinetics of [3H]microcystin-LR in isolated perfused rat livers.

Isolated rat livers were perfused for 60 min with either 0.3 or 0.5 microgram/ml (initial volume, 119 ml) of [3H]microcystin-LR at a constant flow of 10 ml/min in a recirculating system. During the 60-min exposure, toxin caused stimulation of glycogenolysis, liver engorgement, and cessation of bile flow. Electron micrographs of liver showed dilation of bile canaliculi and the space of Disse. loss of sinusoidal lining architecture, and decreased hepatocyte intercellular contacts. Although hepatocytes did not exhibit overt necrosis, mitochondria were hydropic, occasionally encircled by whorls of rough endoplasmic reticulum, and desmosomal tonofilaments were decreased on the plasma membrane lateral surface. Isolated mitochondria displayed inhibition of state 3 respiration and a 50-60% decrease in the respiratory control index, characteristic of hydropism. Distribution of radiolabel was 1.7% to bile, 79% to perfusate, and 16% to liver. Two to four percent was recovered in perfusate that leaked from the surface of the liver. Of the radiolabel found in bile and perfusate, 78 and 100% were associated with parent toxin, respectively. The radiolabel in liver, associated with the cytosolic fraction (S-100), corresponded to parent toxin (15%) and to a more-polar component(s) (85%). The elimination half-life from perfusate was 130 +/- 10 min (0.5 microgram/ml) and the hepatic extraction ratio 0.07 +/- 0.01. Although the calculated hepatic extraction ratio was low, there was a significant accumulation of microcystin in the liver. Many toxic effects of microcystin in the perfused liver mimicked those observed in the whole animal, suggesting that this model can be used as an alternative to whole animals for screening of potential therapeutic agents.

Use of immunoelectron microscopy to show Ebola virus during the 1989 United States epizootic.

A filovirus, serologically related to Ebola virus, was detected by "post-embedment" immunoelectron microscopical examination of MA-104 cells. These had been infected by inoculation with serum samples obtained during the 1989 epizootic in cynomolgus monkeys (Macaca fascicularis), imported from the Philippines and maintained at Reston, Virginia, USA, a primate holding facility. The immunoelectron microscopy method, when used in conjunction with standard transmission electron microscopy (TEM) of infected cells, provided consistent results and was simple to perform in this epizootic. It is concluded that immunoelectron microscopy is potentially useful in the direct immunological diagnosis of Ebola and related filoviral infections (such as Marburg) in clinical samples obtained from those with acute infection.