Background

Ebola virus made headlines in the 1990's as a devastating viral infection, ravaging its victims and leaving them in a hemorrhagic diathesis. Today, it again makes headlines as a potential weapon in bio-terrorist acts. Ebola haemorrhagic fever (EHF) is one of the most virulent viral diseases known to humankind, causing death in 50-90% of all clinically ill cases. The disease has its origins in the jungles of Africa and Asia. The Ebola virus is transmitted by direct contact with the blood, secretions, organs or semen of infected persons. Transmission through semen may occur up to seven weeks after clinical recovery. Transmission of the Ebola virus has also occurred by handling ill or dead infected chimpanzees. Health care workers have frequently been infected while attending patients. Contaminated syringes and needles have also been documented to cause transmission. Incubation is 2 to 21 days. Ebola is often characterized by the sudden onset of fever, weakness, muscle pain, headache and sore throat. This is followed by vomiting, diarrhoea, rash, limited kidney and liver functions, and both internal and external bleeding.

Outline

Epidemiology
Disease Associations
Pathogenesis
Laboratory/Radiologic/Other Diagnostic Testing
Gross Appearance and Clinical Variants
Histopathological Features and Variants
Special Stains/Immunohistochemistry/Electron Microscopy
Differential Diagnosis
Prognosis and Treatment
Commonly Used Terms

EPIDEMIOLOGY	CHARACTERIZATION
GENERAL	Natural reservoir is in the rain forests of Africa and Asia Outbreaks may be secondary to: Rodents Plant virus Mammals
	First identified in a western equatorial province of Sudan and in a nearby region of Zaire (now Democratic Republic of the Congo) in 1976 after significant epidemics in Yambuku, northern Zaire, and Nzara, southern Sudan. Additional outbreaks have been recorded in: Sudan Zaire Gabon Uganda
Serologic survey among hospital and health center workers during the Ebola hemorrhagic fever outbreak in Kikwit, Democratic Republic of the Congo, 1995. Tomori O, Bertolli J, Rollin PE, Fleerackers Y, Guimard Y, De Roo A, Feldmann H, Burt F, Swanepoel R, Killian S, Khan AS, Tshioko K, Bwaka M, Ndambe R, Peters CJ, Ksiazek TG. World Health Organization, Harare, Zimbabwe.	J Infect Dis 1999 Feb;179 Suppl 1:S98-101 Abstract quote From May to July 1995, a serologic and interview survey was conducted to describe Ebola hemorrhagic fever (EHF) among personnel working in 5 hospitals and 26 health care centers in and around Kikwit, Democratic Republic of the Congo. Job-specific attack rates estimated for Kikwit General Hospital, the epicenter of the EHF epidemic, were 31% for physicians, 11% for technicians/room attendants, 10% for nurses, and 4% for other workers. Among 402 workers who did not meet the EHF case definition, 12 had borderline positive antibody test results; subsequent specimens from 4 of these tested negative. Although an old infection with persistent Ebola antibody production or a recent atypical or asymptomatic infection cannot be ruled out, if they occur at all, they appear to be rare. This survey demonstrated that opportunities for transmission of Ebola virus to personnel in health facilities existed in Kikwit because blood and body fluid precautions were not being universally followed.
Prevalence of IgG antibodies to Ebola virus in individuals during an Ebola outbreak, Democratic Republic of the Congo, 1995. Busico KM, Marshall KL, Ksiazek TG, Roels TH, Fleerackers Y, Feldmann H, Khan AS, Peters CJ. Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.	J Infect Dis 1999 Feb;179 Suppl 1:S102-7 Abstract quote During the 1995 outbreak of Ebola (EBO) hemorrhagic fever in Kikwit, Democratic Republic of Congo, two surveys using a new ELISA for EBO (subtype Zaire) virus antigen were conducted to assess the prevalence of EBO IgG antibodies among residents of Kikwit and the surrounding area. The first study determined the proportion of antibody-positive individuals who were self-identified forest and city workers from the Kikwit area. Serum samples from 9 (2.2%) of 414 workers had IgG EBO antibodies. The second study determined the proportion of EBO antibody-positive individuals who lived in villages surrounding Kikwit. The prevalence of IgG EBO antibodies in this population was 9.3% (151161). The difference in the overall prevalence of EBO antibodies may indicate that villagers have a greater chance of exposure to EBO virus compared with those living in and in close proximity to cities.
Ebola between outbreaks: intensified Ebola hemorrhagic fever surveillance in the Democratic Republic of the Congo, 1981-1985. Jezek Z, Szczeniowski MY, Muyembe-Tamfum JJ, McCormick JB, Heymann DL. World Health Organization, Geneva, Switzerland.	J Infect Dis 1999 Feb;179 Suppl 1:S60-4 Abstract quote Surveillance for Ebola hemorrhagic fever was conducted in the Democratic Republic of the Congo from 1981 to 1985 to estimate the incidence of human infection. Persons who met the criteria of one of three different case definitions were clinically evaluated, and blood was obtained for antibody confirmation by IFA. Contacts of each case and 4 age- and sex-matched controls were also clinically examined and tested for immunofluorescent antibody. Twenty-one cases of Ebola infection (persons with an antibody titer of > or = 1:64, or lower if they fit the clinical case definition) were identified, with a maximum 1-year incidence of 9 and a case fatality rate of 43%. Cases occurred throughout the year, but most (48%) occurred early in the rainy season. Fifteen percent of contacts had antibody titers > or =1:64 to Ebola virus, compared with 1% of controls (P < .0001). Results suggest that Ebola virus periodically emerges from nature to infect humans, that person-to-person transmission is relatively limited, and that amplification to large epidemics is unusual.
Epidemiologic and clinical aspects of the Ebola virus epidemic in Mosango, Democratic Republic of the Congo, 1995. Ndambi R, Akamituna P, Bonnet MJ, Tukadila AM, Muyembe-Tamfum JJ, Colebunders R. Mosango General Hospital, Democratic Republic of the Congo.	J Infect Dis 1999 Feb;179 Suppl 1:S8-10 Abstract quote Twenty-three Ebola hemorrhagic fever (EHF) cases (15 males, 8 females) were identified in Mosango, Democratic Republic of the Congo; 18 (78%) of them died. Eight of the patients came from Kikwit General Hospital and were hospitalized at Mosango General Hospital, 10 acquired their infection at the Mosango hospital and were treated there, and 5 acquired their infection through contact with a hospitalized patient but were never hospitalized themselves. For most of the EHF cases, it was clear that they had been in contact with blood or body fluids of another EHF patient. The Ebola outbreak in Mosango remained relatively small, probably because hygienic conditions in this hospital were relatively good at the time of the outbreak and because as soon as the epidemic was recognized, barrier nursing techniques were used.
Ebola and Marburg virus antibody prevalence in selected populations of the Central African Republic. Gonzalez JP, Nakoune E, Slenczka W, Vidal P, Morvan JM. Institut Francais de Recherche Scientifique pour le Developpement en Cooperation IRD-Orstom, Paris, France.	Microbes Infect 2000 Jan;2(1):39-44 Abstract quote With the natural history of the filovirus family seemingly unknown, filovirus ecology in its natural environment remains a rudimentary field of research. In order to investigate the maintenance cycle of filovirus in Central Africa, a study was conducted within the rain forest of the Central African Republic. The epidemiological study determines the frequency and distribution of filovirus seroprevalence in a selected human population. Using an ELISA, serum samples from Pygmy and non-Pygmy populations were tested for Ebola-Zaire virus and Marburg (MBG) virus antibody. Filovirus antibody reacting sera were found in all zones investigated, and in all populations studied (Ebola virus IgG 5.3%; Marburg virus IgG 2.4%). Pygmies appeared to have a significantly higher seroprevalence (P < 0.03) against Ebola-Zaire virus (7.02%) than non-Pygmies (4.2%). MBG virus or related unknown filovirus strains also seem to be present in the western part of Central Africa. MBG virus antibodies were present in different Pygmy groups (ranging from 0.7 to 5.6%, mean 2.05%) and in several non-Pygmy populations (ranging from 0.0 to 3.9%, mean 3.4%) without an overall significant difference between the two groups (P = 0.14). The potentialities of nonpathogenic filovirus strains circulating in the Central African Republic are discussed.
Unexpected Ebola virus in a tertiary setting: clinical and epidemiologic aspects. Richards GA, Murphy S, Jobson R, Mer M, Zinman C, Taylor R, Swanepoel R, Duse A, Sharp G, De La Rey IC, Kassianides C. Department of Pulmonology and Critical Care, Johannesburg Hospital, and University of the Witwatersrand, South Africa.	Crit Care Med 2000 Jan;28(1):240-4 Abstract quote OBJECTIVES: To describe the clinical manifestations of viral hemorrhagic fever, and to increase clinicians' awareness and knowledge of these illnesses. DESIGN: Retrospective study of the clinical and laboratory data and management of two cases of Ebola virus infection with key epidemiologic data provided. SETTING: Two tertiary care hospitals. PATIENTS: Two adult patients, the index case and the source patient, both identified as having Ebola, one of whom originated in Gabon. INTERVENTIONS: One patient was admitted to the intensive care unit. The other was managed in a general ward. MEASUREMENT AND MAIN RESULTS: Clinical and laboratory data are reported. One patient, a healthcare worker who contracted this illness in the course of her work, died of refractory thrombocytopenia and an intracerebral bleed. The source patient survived. Despite a long period during which the diagnosis was obscure, none of the other 300 contacts contracted the illness. CONCLUSIONS: Identification of high-risk patients and use of universal blood and body fluid precautions will considerably decrease the risk of nosocomial spread of viral hemorrhagic fevers.

 

DISEASE ASSOCIATIONS

CHARACTERIZATION

Leptospirosis and Ebola virus infection in five gold-panning villages in northeastern Gabon. Bertherat E, Renaut A, Nabias R, Dubreuil G, Georges-Courbot MC. Centre International de Recherches Medicales de Franceville, Gabon.

Am J Trop Med Hyg 1999 Apr;60(4):610-5 Abstract quote An exhaustive epidemiologic and serologic survey was carried out in five gold-panning villages situated in northeastern Gabon to estimate the degree of exposure of to leptospirosis and Ebola virus. The seroprevalence was 15.7% for leptospirosis and 10.2% for Ebola virus. Sixty years after the last seroepidemiologic survey of leptospirosis in Gabon, this study demonstrates the persistence of this infection among the endemic population and the need to consider it as a potential cause of hemorrhagic fever in Gabon. There was no significant statistical correlation between the serologic status of populations exposed to both infectious agents, indicating the lack of common risk factors for these diseases.

 

PATHOGENESIS	CHARACTERIZATION
VIRAL STRAINS
Emergence of subtype Zaire Ebola virus in Gabon. Volchkov V, Volchkova V, Eckel C, Klenk HD, Bouloy M, LeGuenno B, Feldmann H. Institut fur Virologie, Philipps-Universitat, Marburg, Germany.	Virology 1997 May 26;232(1):139-44 Abstract quote Gabon has recently been struck three times by Ebola hemorrhagic fever. The first isolate originating from the 1994 outbreak has been subjected to molecular characterization of its GP and VP24 genes. Sequence analysis demonstrates that the agent, Gabon-94 virus, belongs to subtype Zaire of Ebola virus. The isolate is closely related to the Kikwit-95 isolate, and both viruses seem to have evolved from a progenitor virus different from that of the Zaire-76 isolates. The relatively close relationship of all subtype Zaire viruses isolated at different geographical locations and up to 20 years apart suggests an extreme conservation in the yet unknown natural reservoir of Ebola viruses. The level of genetic variability in the human host might be different as indicated by the comparison of isolates from a single outbreak (Mayinga-76 and Eckron-76), but needs further investigation on clinical material of patients by PCR since both isolates have different levels of passages in tissue culture.
Epidemiology of Ebola (subtype Reston) virus in the Philippines, 1996. Miranda ME, Ksiazek TG, Retuya TJ, Khan AS, Sanchez A, Fulhorst CF, Rollin PE, Calaor AB, Manalo DL, Roces MC, Dayrit MM, Peters CJ. Research Institute for Tropical Medicine, and Field Epidemiology Training Program, Department of Health, Manila, Philippines	J Infect Dis 1999 Feb;179 Suppl 1:S115-9 Abstract quote Ebola (subtype Reston [EBO-R]) virus infection was detected in macaques imported into the United States from the Philippines in March 1996. Studies were initiated in the Philippines to identify the source of the virus among monkey-breeding and export facilities, to establish surveillance and testing, and to assess the risk and significance of EBO-R infections in humans who work in these facilities. Over a 5-month period, acutely infected animals were found at only one facility, as determined using Ebola antigen detection. Three of 1732 monkeys and 1 of 246 animal handlers tested had detectable antibodies; all were from the same facility, which was the source of infected monkeys imported to the United States. Virus transmission, which was facilitated by poor infection-control practices, continued for several months in one facility and was stopped only when the facility was depopulated. None of the 246 employees of the facilities or 4 contacts of previously antibody-positive individuals reported an Ebola-like illness. This investigation suggests that human EBO-R infection is rare.
PATHOGENIC MECHANISMS
A novel hypothesis to explain the hemorrhagic and connective tissue manifestations of Ebola virus infection. Tilson MD, Ozsvath KJ, Hirose H, Xia S, Lahita R. St. Luke's/Roosevelt Hospital Center, Columbia University, New York 10027, USA	Clin Immunol Immunopathol 1996 Dec;81(3):303-6 Abstract quote The hemorrhagic and connective tissue complications of infection with Ebola virus are poorly understood. While searching for homologies and motifs of the aortic aneurysm-associated autoantigenic protein 40 kDa (AAAP-40), we have noted some short sequences (possibly shared epitopes) that occur in the envelope glycoprotein (40 kDa) of the Ebola virus. As a first step toward determining whether molecular mimicry of human matrix proteins by the Ebola virus protein might explain some of the severe connective tissue manifestations of infection, we have tested whether immunoglobulin (IgG) purified from the sera of patients with abdominal aortic aneurysm (AAA) are immunoreactive with the 40-kDa protein of the Ebola virus. Immunoblots of soluble Ebola proteins (strain Mayinga/Zaire) were probed with IgG's purified from the sera of eight patients with AAA and two healthy young control volunteers. The proteins were also probed with IgG extracted from the walls of two surgical aneurysm specimens. Serum IgG from eight consecutively studied AAA patients was immunoreactive with an Ebola virus protein of 40 kDa, consistent with the envelope glycoprotein. IgG's extracted from the walls of two AAAs were also reactive. The control sera were not reactive. In addition to the Ebola sequences in AAAP-40, an Ebola sequence also occurs in the microfibril-associated glycoprotein-4 (MAGP-4), which is distributed ubiquitously throughout connective tissue with elastin. We hypothesize that the catastrophic hemorrhagic and connective tissue complications of Ebola virus infection may be the result of these shared epitopes.
Apoptosis in fatal Ebola infection. Does the virus toll the bell for immune system? Baize S, Leroy EM, Mavoungou E, Fisher-Hoch SP. Centre International de Recherches Medicales de Franceville, Gabon, France.	Apoptosis 2000 Feb;5(1):5-7 Abstract quote In fatal Ebola virus hemorrhagic fever massive intravascular apoptosis develops rapidly following infection and progressing relentlessly until death. While data suggest that T lymphocytes are mainly deleted by apoptosis in PBMC of human fatal cases, experimental Ebola infection in animal models have shown some evidence of destruction of lymphocytes in spleen and lymph nodes probably involving both T and B cells. Nevertheless, we are able to conclude from the accumulated evidence that early interactions between Ebola virus and the immune system, probably via macrophages, main targets for viral replication, lead to massive destruction of immune cells in fatal cases.
The pathogenesis of Ebola hemorrhagic fever. Takada A, Kawaoka Y. Division of Virology, Dept of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 108-8639, Tokyo, Japan	Trends Microbiol 2001 Oct;9(10):506-11 Abstract quote Ebola virus causes lethal hemorrhagic disease in humans, yet there are still no satisfactory biological explanations to account for its extreme virulence. This review focuses on recent findings relevant to understanding the pathogenesis of Ebola virus infection and developing vaccines and effective therapy. The available data suggest that the envelope glycoprotein and the interaction of some viral proteins with the immune system are likely to play important roles in the extraordinary pathogenicity of this virus. There are also indications that genetically engineered vaccines, including plasmid DNA and viral vectors expressing Ebola virus proteins, and passive transfer of neutralizing antibodies could be feasible options for the control of Ebola virus-associated disease.
HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress. Martin-Serrano J, Zang T, Bieniasz PD. Aaron Diamond AIDS Research Center and The Rockefeller University, New York, New York, USA..	Nat Med 2001 Dec;7(12):1313-9 Abstract quote Retroviral Gag proteins encode sequences, termed late domains, which facilitate the final stages of particle budding from the plasma membrane. We report here that interactions between Tsg101, a factor involved in endosomal protein sorting, and short peptide motifs in the HIV-1 Gag late domain and Ebola virus matrix (EbVp40) proteins are essential for efficient egress of HIV-1 virions and Ebola virus-like particles. EbVp40 recruits Tsg101 to sites of particle assembly and a short, EbVp40-derived Tsg101-binding peptide sequence can functionally substitute for the HIV-1 Gag late domain. Notably, recruitment of Tsg101 to assembling virions restores budding competence to a late-domain-defective HIV-1 in the complete absence of viral late domain. These studies define an essential virus-host interaction that is conserved in two unrelated viruses. Because the Tsg101 is recruited by small, conserved viral sequence motifs, agents that mimic these structures are potential inhibitors of the replication of these lethal human pathogens.
Early immune responses accompanying human asymptomatic Ebola infections. Leroy EM, Baize S, Debre P, Lansoud-Soukate J, Mavoungou E. Centre International de Recherches Medicales de Franceville, BP 769, Franceville, Gabon.	Clin Exp Immunol 2001 Jun;124(3):453-60 Abstract quote In a recent study we identified certain asymptomatic individuals infected by Ebola virus (EBOV) who mounted specific IgG and early and strong inflammatory responses. Here, we further characterized the primary immune response to EBOV during the course of asymptomatic infection in humans. Inflammatory responses occurred in temporal association with anti-inflammatory phase composed by soluble antagonist IL-1RA, circulating TNF receptors, IL-10 and cortisol. At the end of the inflammatory process, mRNA expression of T-cell cytokines (IL-2 and IL-4) and activation markers (CD28, CD40L and CTLA4) was up-regulated, strongly suggesting T-cell activation. This T-cell activation was followed by EBOV-specific IgG responses (mainly IgG3 ang IgG1), and by marked and sustained up-regulation of IFN gamma, FasL and perforin mRNA expression, suggesting activation of cytotoxic cells. The terminal down-regulation of these latter markers coincided with the release of the apoptotic marker 41/7 NMP in blood and with the disappearance of viral RNA from PBMC, suggesting that infected cells are eliminated by cytotoxic mechanisms. Finally, RT-PCR analysis of TCR-V beta repertoire usage showed that TCR-V beta 12 mRNA was never expressed during the infection. Taken together, these findings improve our understanding about immune response during human asymptomatic Ebola infection, and throw new light on protection against Ebola virus.
Folate receptor-alpha is a cofactor for cellular entry by Marburg and Ebola viruses. Chan SY, Empig CJ, Welte FJ, Speck RF, Schmaljohn A, Kreisberg JF, Goldsmith MA. Gladstone Institute of Virology and Immunology, San Francisco, CA 94141, USA.	Cell 2001 Jul 13;106(1):117-26 Abstract quote Human infections by Marburg (MBG) and Ebola (EBO) viruses result in lethal hemorrhagic fever. To identify cellular entry factors employed by MBG virus, noninfectible cells transduced with an expression library were challenged with a selectable pseudotype virus packaged by MBG glycoproteins (GP). A cDNA encoding the folate receptor-alpha (FR-alpha) was recovered from cells exhibiting reconstitution of viral entry. A FR-alpha cDNA was recovered in a similar strategy employing EBO pseudotypes. FR-alpha expression in Jurkat cells facilitated MBG or EBO entry, and FR-blocking reagents inhibited infection by MBG or EBO. Finally, FR-alpha bound cells expressing MBG or EBO GP and mediated syncytia formation triggered by MBG GP. Thus, FR-alpha is a significant cofactor for cellular entry for MBG and EBO viruses.
Ebola virus VP40-induced particle formation and association with the lipid bilayer. Jasenosky LD, Neumann G, Lukashevich I, Kawaoka Y. Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.	J Virol 2001 Jun;75(11):5205-14 Abstract quote Viral protein 40 (VP40) of Ebola virus appears equivalent to matrix proteins of other viruses, yet little is known about its role in the viral life cycle. To elucidate the functions of VP40, we investigated its ability to induce the formation of membrane-bound particles when it was expressed apart from other viral proteins. We found that VP40 is indeed able to induce particle formation when it is expressed in mammalian cells, and this process appeared to rely on a conserved N-terminal PPXY motif, as mutation or loss of this motif resulted in markedly reduced particle formation. These findings demonstrate that VP40 alone possesses the information necessary to induce particle formation, and this process most likely requires cellular WW domain-containing proteins that interact with the PPXY motif of VP40. The ability of VP40 to bind cellular membranes was also studied. Flotation gradient analysis indicated that VP40 binds to membranes in a hydrophobic manner, as NaCl at 1 M did not release the protein from the lipid bilayer. Triton X-114 phase-partitioning analysis suggested that VP40 possesses only minor features of an integral membrane protein. We confirmed previous findings that truncation of the 50 C-terminal amino acids of VP40 results in decreased association with cellular membranes and demonstrated that this deletion disrupts hydrophobic interactions of VP40 with the lipid bilayer, as well as abolishing particle formation. Truncation of the 150 C-terminal amino acids or 100 N-terminal amino acids of VP40 enhanced the protein's hydrophobic association with cellular membranes. These data suggest that VP40 binds the lipid bilayer in an efficient yet structurally complex fashion.
Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity. Volchkov VE, Volchkova VA, Muhlberger E, Kolesnikova LV, Weik M, Dolnik O, Klenk HD. Institut fur Virologie, Philipps-Universitat, Robert-Koch-Strasse 17, 35037 Marburg, Germany.	Science 2001 Mar 9;291(5510):1965-9 Abstract quote To study the mechanisms underlying the high pathogenicity of Ebola virus, we have established a system that allows the recovery of infectious virus from cloned cDNA and thus permits genetic manipulation. We created a mutant in which the editing site of the gene encoding envelope glycoprotein (GP) was eliminated. This mutant no longer expressed the nonstructural glycoprotein sGP. Synthesis of GP increased, but most of it accumulated in the endoplasmic reticulum as immature precursor. The mutant was significantly more cytotoxic than wild-type virus, indicating that cytotoxicity caused by GP is down-regulated by the virus through transcriptional RNA editing and expression of sGP.

 

LABORATORY/RADIOLOGIC/ OTHER TESTS	CHARACTERIZATION
RADIOLOGIC
LABORATORY MARKERS
ELISA for the detection of antibodies to Ebola viruses. Ksiazek TG, West CP, Rollin PE, Jahrling PB, Peters CJ. Disease Assessment Division, US Army Medical Research Institute of Infectious Disease, Fort Detrick, Frederick, Maryland, USA	J Infect Dis 1999 Feb;179 Suppl 1:S192-8 Abstract quote EIAs for IgG and IgM antibodies directed against Ebola (EBO) viral antigens have been developed and evaluated using sera of animals and humans surviving infection with EBO viruses. The IgM capture assay detected anti-EBO (subtype Reston) antibodies in the sera of 5 of 5 experimentally infected animals at the time they succumbed to lethal infections. IgM antibodies were also detected in the serum of a human who was infected with EBO (subtype Reston) during a postmortem examination of an infected monkey. The antibody was detectable as early as day 6 after infection in experimentally infected animals and persisted for <90 days. The IgG response was less rapid; however, it persisted for >400 days in 3 animals who survived infection, and it persisted for approximately 10 years after infection in the sera of 2 humans. Although these data are limited by the number of sera available for verification, the IgM assay seems to have great promise as a diagnostic tool. Furthermore the long-term persistence of the IgG antibodies measured by this test strongly suggests that the ELISA will be useful in field investigations of EBO virus.
Development and Evaluation of a Fluorogenic 5' Nuclease Assay To Detect and Differentiate between Ebola Virus Subtypes Zaire and Sudan. Gibb TR, Norwood DA Jr, Woollen N, Henchal EA. Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland 21702-5011.	J Clin Microbiol 2001 Nov;39(11):4125-30 Abstract quote The ability to rapidly recognize Ebola virus infections is critical to quickly limit further spread of the disease. A rapid, sensitive, and specific laboratory diagnostic test is needed to confirm outbreaks of Ebola virus infection and to distinguish it from other diseases that can cause similar clinical symptoms. A one-tube reverse transcription-PCR assay for the identification of Ebola virus subtype Zaire (Ebola Zaire) and Ebola virus subtype Sudan (Ebola Sudan) was developed and evaluated by using the ABI PRISM 7700 sequence detection system. This assay uses one common primer set and two differentially labeled fluorescent probes to simultaneously detect and differentiate these two subtypes of Ebola virus. The sensitivity of the primer set was comparable to that of previously designed primer sets, as determined by limit-of-detection experiments. This assay is unique in its ability to simultaneously detect and differentiate Ebola Zaire and Ebola Sudan. In addition, this assay is compatible with emerging rapid nucleic acid analysis platforms and therefore may prove to be a very useful diagnostic tool for the control and management of future outbreaks.
Detection of ebola viral antigen by enzyme-linked immunosorbent assay using a novel monoclonal antibody to nucleoprotein. Niikura M, Ikegami T, Saijo M, Kurane I, Miranda ME, Morikawa S. Department of Virology 1, National Institute of Infectious Diseases, The University of Tokyo, Tokyo, Japan.	J Clin Microbiol 2001 Sep;39(9):3267-71 Abstract quote With the increase in international traffic, the risk of introducing rare but severe infectious diseases like Ebola hemorrhagic fever is increasing all over the world. However, the system for the diagnosis of Ebola virus infection is available in a limited number of countries. In the present study, we developed an Ebola virus antigen-detection enzyme-linked immunosorbent assay (ELISA) system using a novel monoclonal antibody (MAb) to the nucleoprotein (NP). This antibody recognized an epitope defined by a 26-amino-acid stretch near the C terminus of NP. In a sandwich ELISA system with the MAb, as little as 30 ng of purified recombinant NP (rNP) was detected. Although this MAb was prepared by immunization with rNP of subtype Zaire, it also reacted to the corresponding region of NP derived from the Reston and Sudan subtypes. These results suggest that our ELISA system should work with three of four Ebola subtypes. Furthermore, our ELISA system detected the NP in subtype Reston-infected monkey specimens, while the background level in noninfected specimens was very low, suggesting the usefulness of the ELISA for laboratory diagnosis with clinical specimens.

 

GROSS APPEARANCE/ CLINICAL VARIANTS	CHARACTERIZATION
GENERAL
Clinical, virologic, and immunologic follow-up of convalescent Ebola hemorrhagic fever patients and their household contacts, Kikwit, Democratic Republic of the Congo. Commission de Lutte contre les Epidemies a Kikwit. Rowe AK, Bertolli J, Khan AS, Mukunu R, Muyembe-Tamfum JJ, Bressler D, Williams AJ, Peters CJ, Rodriguez L, Feldmann H, Nichol ST, Rollin PE, Ksiazek TG. Epidemiology Program Office and Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, USA.	J Infect Dis 1999 Feb;179 Suppl 1:S28-35 Abstract quote A cohort of convalescent Ebola hemorrhagic fever (EHF) patients and their household contacts (HHCs) were studied prospectively to determine if convalescent body fluids contain Ebola virus and if secondary transmission occurs during convalescence. Twenty-nine EHF convalescents and 152 HHCs were monitored for up to 21 months. Blood specimens were obtained and symptom information was collected from convalescents and their HHCs; other body fluid specimens were also obtained from convalescents. Arthralgias and myalgia were reported significantly more often by convalescents than HHCs. Evidence of Ebola virus was detected by reverse transcription-polymerase chain reaction in semen specimens up to 91 days after disease onset; however, these and all other non-blood body fluids tested negative by virus isolation. Among 81 initially antibody negative HHCs, none became antibody positive. Blood specimens of 5 HHCs not identified as EHF patients were initially antibody positive. No direct evidence of convalescent-to-HHC transmission of EHF was found, although the semen of convalescents may be infectious. The existence of initially antibody-positive HHCs suggests that mild cases of Ebola virus infection occurred and that the full extent of the EHF epidemic was probably underestimated.
VARIANTS
Late ophthalmologic manifestations in survivors of the 1995 Ebola virus epidemic in Kikwit, Democratic Republic of the Congo. Kibadi K, Mupapa K, Kuvula K, Massamba M, Ndaberey D, Muyembe-Tamfum JJ, Bwaka MA, De Roo A, Colebunders R. Kikwit General Hospital, Democratic Republic of the Congo	J Infect Dis 1999 Feb;179 Suppl 1:S13-4 Abstract quote Three (15%) of 20 survivors of the 1995 Ebola outbreak in the Democratic Republic of the Congo enrolled in a follow-up study and 1 other survivor developed ocular manifestations after being asymptomatic for 1 month. Patients complained of ocular pain, photophobia, hyperlacrimation, and loss of visual acuity. Ocular examination revealed uveitis in all 4 patients. All patients improved with a topical treatment of 1% atropine and steroids.

 

HISTOLOGICAL TYPES	CHARACTERIZATION
GENERAL
VARIANTS
Pathology of experimental Ebola virus infection in African green monkeys. Involvement of fibroblastic reticular cells. Davis KJ, Anderson AO, Geisbert TW, Steele KE, Geisbert JB, Vogel P, Connolly BM, Huggins JW, Jahrling PB, Jaax NK. Pathology Division, USAMRIID, Ft Detrick, MD 21702-5011, USA. B	Arch Pathol Lab Med 1997 Aug;121(8):805-19 Abstract quote 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.

 

SPECIAL STAINS/IMMUNOPEROXIDASE/ OTHER	CHARACTERIZATION
SPECIAL STAINS
IMMUNOPEROXIDASE
A novel immunohistochemical assay for the detection of Ebola virus in skin: implications for diagnosis, spread, and surveillance of Ebola hemorrhagic fever. Commission de Lutte contre les Epidemies a Kikwit. Zaki SR, Shieh WJ, Greer PW, Goldsmith CS, Ferebee T, Katshitshi J, Tshioko FK, Bwaka MA, Swanepoel R, Calain P, Khan AS, Lloyd E, Rollin PE, Ksiazek TG, Peters CJ. Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.	J Infect Dis 1999 Feb;179 Suppl 1:S36-47 Abstract quote Laboratory diagnosis of Ebola hemorrhagic fever (EHF) is currently performed by virus isolation and serology and can be done only in a few high-containment laboratories worldwide. In 1995, during the EHF outbreak in the Democratic Republic of Congo, the possibility of using immunohistochemistry (IHC) testing of formalin-fixed postmortem skin specimens was investigated as an alternative diagnostic method for EHF. Fourteen of 19 cases of suspected EHF met the surveillance definition for EHF and were positive by IHC. IHC, serologic, and virus isolation results were concordant for all EHF and non-EHF cases. IHC and electron microscopic examination showed that endothelial cells, mononuclear phagocytes, and hepatocytes are main targets of infection, and IHC showed an association of cellular damage with viral infection. The finding of abundant viral antigens and particles in the skin of EHF patients suggests an epidemiologic role for contact transmission. IHC testing of formalin-fixed skin specimens is a safe, sensitive, and specific method for laboratory diagnosis of EHF and should be useful for EHF surveillance and prevention.
ELECTRON MICROSCOPY
Differentiation of filoviruses by electron microscopy. Geisbert TW, Jahrling PB. Pathology Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD 21702-5011, USA.	Virus Res 1995 Dec;39(2-3):129-50 Abstract quote 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.

 

DIFFERENTIAL DIAGNOSIS	KEY DIFFERENTIATING FEATURES
Marburg virus	Produces essentially identical clinical disease

 

PROGNOSIS AND TREATMENT	CHARACTERIZATION
PROGNOSTIC FACTORS
PROPHYLAXIS	Primary mode of person-to-person transmission is contact with contaminated blood, secretions or body fluid Any person who has had close physical contact with patients should be kept under strict surveillance, i.e. body temperature checks twice a day, with immediate hospitalization and strict isolation recommended in case of temperatures above 38.3°C (101°F). Casual contacts should be placed on alert and asked to report any fever Surveillance of suspected cases should continue for three weeks after the date of their last contact. Hospital personnel who come into close contact with patients or contaminated materials without barrier nursing attire must be considered exposed and put under close supervised surveillance.
TREATMENT
Ebola virus can be effectively neutralized by antibody produced in natural human infection. Maruyama T, Rodriguez LL, Jahrling PB, Sanchez A, Khan AS, Nichol ST, Peters CJ, Parren PW, Burton DR. Departments of Immunology and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.	J Virol 1999 Jul;73(7):6024-30 Abstract quote The activity of antibodies against filoviruses is poorly understood but has important consequences for vaccine design and passive prophylaxis. To investigate this activity, a panel of recombinant human monoclonal antibodies to Ebola virus antigens was isolated from phage display libraries constructed from RNA from donors who recovered from infection in the 1995 Ebola virus outbreak in Kikwit, Democratic Republic of Congo. Antibodies reactive with nucleoprotein (NP), envelope glycoprotein (GP), and secreted envelope glycoprotein (sGP) were characterized by immunofluorescence and radioimmunoprecipitation assays. Four antibodies reacting strongly with sGP and weakly with GP and two antibodies reacting with NP were not neutralizing. An antibody specific for GP neutralized Ebola virus to 50% at 0.4 microgram/ml as the recombinant Fab fragment and to 50% at 0.3 microgram/ml (90% at 2.6 microgram/ml) as the corresponding whole immunoglobulin G1 molecule. The studies indicate that neutralizing antibodies are produced in infection by Ebola virus although probably at a relatively low frequency. The neutralizing antibody may be useful in vaccine design and as a prophylactic agent against Ebola virus infection.
Individual and Bivalent Vaccines Based on Alphavirus Replicons Protect Guinea Pigs against Infection with Lassa and Ebola Viruses. Pushko P, Geisbert J, Parker M, Jahrling P, Smith J. Virology Division, United States Army Medical Research Institute for Infectious Diseases, Fort Detrick, Frederick, Maryland.	J Virol 2001 Dec;75(23):11677-85 Abstract quote Lassa and Ebola viruses cause acute, often fatal, hemorrhagic fever diseases, for which no effective vaccines are currently available. Although lethal human disease outbreaks have been confined so far to sub-Saharan Africa, they also pose significant epidemiological concern worldwide as demonstrated by several instances of accidental importation of the viruses into North America and Europe. In the present study, we developed experimental individual vaccines for Lassa virus and bivalent vaccines for Lassa and Ebola viruses that are based on an RNA replicon vector derived from an attenuated strain of Venezuelan equine encephalitis virus. The Lassa and Ebola virus genes were expressed from recombinant replicon RNAs that also encoded the replicase function and were capable of efficient intracellular self-amplification. For vaccinations, the recombinant replicons were incorporated into virus-like replicon particles. Guinea pigs vaccinated with particles expressing Lassa virus nucleoprotein or glycoprotein genes were protected from lethal challenge with Lassa virus. Vaccination with particles expressing Ebola virus glycoprotein gene also protected the animals from lethal challenge with Ebola virus. In order to evaluate a single vaccine protecting against both Lassa and Ebola viruses, we developed dual-expression particles that expressed glycoprotein genes of both Ebola and Lassa viruses. Vaccination of guinea pigs with either dual-expression particles or with a mixture of particles expressing Ebola and Lassa virus glycoprotein genes protected the animals against challenges with Ebola and Lassa viruses. The results showed that immune responses can be induced against multiple vaccine antigens coexpressed from an alphavirus replicon and suggested the possibility of engineering multivalent vaccines based upon alphavirus vectors for arenaviruses, filoviruses, and possibly other emerging pathogens.
Vaccine potential of Ebola virus VP24, VP30, VP35, and VP40 proteins. Wilson JA, Bray M, Bakken R, Hart MK. Virology Division, United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Fort Detrick, Frederick, Maryland 21702-5011, USA.	Virology 2001 Aug 1;286(2):384-90 Abstract quote Previous vaccine efforts with Ebola virus Zaire (EBOV-Z) emphasized the potential protective efficacies of immune responses to the surface glycoprotein and the nucleoprotein. To determine whether the VP24, VP30, VP35, and VP40 proteins are also capable of eliciting protective immune responses, these genes were expressed from alphavirus replicons and used to vaccinate BALB/c and C57BL/6 mice. Although all of the VP proteins were capable of inducing protective immune responses, no single VP protein protected both strains of mice tested. VP24, VP30, and VP40 induced protective immune responses in BALB/c mice, whereas C57BL/6 mice survived challenge only after vaccination with VP35. Passive transfer of immune sera to the VP proteins did not protect unvaccinated mice from lethal disease. The demonstration that the VP proteins are capable of eliciting protective immune responses to EBOV-Z indicates that they may be important components of a vaccine designed to protect humans from Ebola hemorrhagic fever.

Henry JB. Clinical Diagnosis and Management by Laboratory Methods. Twentieth Edition. WB Saunders. 2001.
Robbins Pathologic Basis of Disease. Sixth Edition. WB Saunders 1999.

Virus

Last Updated 12/10/2001