Table of Contents:

• Taxonomy Information
• Lifecycle Information
• Genome Information
• Biosafety Information
• Culturing Information
• Epidemiology Information
• Diagnostic Tests Information
• Infected Hosts Information
• Phinet: Pathogen-Host Interaction Network
• Lab Animal Pathobiology & Management
• References
• Data Provenance and Curators

1. Species:
   1. Marburg virus (Website 1):
      1. Common Name: Marburg virus
      2. GenBank Taxonomy No.: 11269
      3. Description: Filoviruses are classified in the order Mononegavirales which also contains the nonsegmented negative-strand RNA virus families Paramyxoviridae, Rhabdoviridae, and Bornaviridae. Members of the family Filoviridae include Marburg virus, a unique agent without known subtypes, and Ebola virus, which has four subtypes, Zaire, Sudan, Reston, and Ivory Coast(Beer et al., 1999).
      4. Variant(s):
         • Marburg virus (strain Musoke) (Website 2):
           • Common Name: Marburg virus (strain Musoke)
           • GenBank Taxonomy No.: 33727
           • Parents: Marburg virus
           • Description: The Musoke strain was isolated in 1980 in Kenya and subsequently purified from an infected Vero cell culture(Beer et al., 1999).
         • Marburg virus (strain Popp) (Website 4):
           • Common Name: Marburg virus (strain Popp)
           • GenBank Taxonomy No.: 33728
           • Parents: Marburg virus
           • Description: The Popp strain was obtained in 1967 during the first filovirus outbreak from the blood of infected guinea pigs(Beer et al., 1999).

1. Marburg Virus Information
   1. Stage Information:
      1. Virion:
         • Size: Marburg and Ebola viruses are pleomorphic particles which vary greatly in length, but the unit length associated with peak infectivity is 790 nm for Marburg virus and 970 nm for Ebola virus.
         • Shape: The virions appear as either long filamentous (and sometimes branched) forms or in shorter U-shaped, 6-shaped (mace-shaped), or circular (ring) configurations. Virions have a uniform diameter of 80 nm and a density of 1.14 g/ml.

1. Genome of Marburg virus
   1. Description: Filoviruses are enveloped, nonsegmented negative-stranded RNA viruses. The two species, Marburg and Ebola virus, are serologically, biochemically, and genetically distinct(Beer et al., 1999).
   2. Single Chromosome(Website 6)
      1. GenBank Accession Number: NC_001608
      2. Size: 19112 bp(Website 6).
      3. Gene Count: 7 genes(Website 6).
      4. Description: The nonsegmented negative-strand RNA genomes of filoviruses show the gene arrangement 3'-NP-VP35-VP40-GP-VP30-VP24-L-5' with a total molecular length of approximately 19 kb. They are the largest known genomes for negative-strand RNA viruses(Beer et al., 1999).

1. Biosafety information for Marburg virus
   1. Level: Because of their aerosol infectivity, high mortality rate, potential for person-to-person transmission, and the lack of commercially available vaccines and chemotherapy, Marburg and Ebola viruses are classified as biosafety level four pathogens(Beer et al., 1999).
   2. Precautions: The patient should be isolated in a single room with an adjoining anteroom serving as its only entrance. The anteroom should contain supplies for routine patient care, as well as gloves, gowns, and masks for the staff. The Appendix lists suggested supplies for the anteroom. Hand-washing facilities should be available in the anteroom, as well as containers of decontaminating solutions. If possible, the patient's room should be at negative air pressure compared with the anteroom and the outside hall, and the air should not be recirculated. However, this is not absolutely required, and does not constitute a reason to transfer the patient. If a room such as described is not available, use adjacent rooms to provide safe and adequate space(MMWR, 1988). Strict barrier-nursing techniques should be enforced: all persons entering the patient's room should wear disposable gloves, gowns, masks, and shoe covers. Protective eye wear should be worn by persons dealing with disoriented or uncooperative patients or performing procedures that might involve the patient's vomiting or bleeding (for example, inserting a nasogastric tube or an intravenous or arterial line). Protective clothing should be donned and removed in the anteroom. Only essential medical and nursing personnel should enter the patient's room and anteroom. Isolation signs listing necessary precautions should be posted outside the anteroom(MMWR, 1988). Lipid-containing viruses, including the enveloped viruses, are among the most readily inactivated of all viral agents. Suitable disinfectant solutions include 0.5% sodium hypochlorite (10% aqueous solution of household bleach), as well as fresh, correctly prepared solutions of glutaraldehyde (2% or as recommended by the manufacturer) and phenolic disinfectants (0.5%-3%). Soaps and detergents can also inactivate these viruses and should be used liberally(MMWR, 1988). Laboratory personnel accidentally exposed to potentially-infected material (for example, through injections or cuts or abrasions on the hands) should immediately wash the infected part, apply a disinfectant solution such as hypochlorite solution, and notify the patient's physician. The person should then be considered as a high-risk contact and placed under surveillance. Accidental spills of potentially contaminated material should be liberally covered with disinfectant solution, left to soak for 30 minutes, and wiped up with absorbent material soaked in disinfectant(MMWR, 1988).
   3. Disposal: The patient should use a chemical toilet. All secretions, excretions, and other body fluids (other than laboratory specimens) should be treated with disinfectant solution. All material used for patients, such as disposable linen and pajamas, should be double-bagged in airtight bags. The outside bags should be sponged with disinfectant solution and later incinerated or autoclaved. Disposable items worn by staff, such as gowns, gloves, etc., should be similarly treated. Disposable items used in patient care (suction catheters, dressings, etc.) should be placed in a rigid plastic container of disinfectant solution. The outside of the container should be sponged with disinfectant, and the container should be autoclaved, incinerated, or otherwise safely discarded(MMWR, 1988). All unnecessary handling of the body, including embalming, should be avoided. Persons who dispose of the corpse must take the same precautions outlined for medical and laboratory staff. The corpse should be placed in an airtight bag and cremated or buried immediately(MMWR, 1988). Disposable items, such as pipette tips, specimen containers, swabs, etc., should be placed in a container filled with disinfectant solution and incinerated. Clothes and blankets that were used by the patient should be washed in a disinfectant, such as hypochlorite solution. Nondisposable items such as endoscopes used in patient care must be cleaned with decontaminating fluids (for example, gluteraldehyde or hypochlorite). Laboratory equipment must be treated similarly. All non-disposable materials that withstand autoclaving should be autoclaved, after they have been soaked in disinfectant solution. The patient's bed and other exposed surfaces in the hospital room, or in vehicles used to transport the patient, should be decontaminated with disinfectant solution(MMWR, 1988).

1. Virus Isolation in Cell Culture :
   1. Description: Virus isolation in cell culture. Acute sera or postmortem tissues are usually positive. Virus growth is detected by cytopathic effect or more usually by fluorescent antibody detection of antigen in cells. It is usually efficient. May require several days, multiple cell systems, and blind passage. BSL-4 laboratory is required. Isolation in cell culture or animals is needed to study the virus, regardless of how it was detected intially(Peters et al., 1996).
2. Vero E6 Cell culture :
   1. Description: In this study they used the Musoke strain of Marburg virus (MBGV), the Mayinga strain of the Zaire species of EBOV (EBOV-Zaire), and the Reston strain of the Reston species of EBOV (EBOV-Reston). Virus stocks were freshly prepared in Vero E6 cells (ATCC 1568). Harvesting was performed when no obvious cytopathic effect was seen. Mock-infected Vero E6 cells were treated the same way in order to prepare a control (mock stock). Vero E6 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum), penicillin (100 U/ml), streptomycin (100 ug/ml), and L-glutamine (2 mM). For virus propagation, DMEM with 2% fetal calf serum was used(Stroher et al., 2001).
   2. Medium: Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum), penicillin (100 U/ml), streptomycin (100 ug/ml), and L-glutamine (2 mM)(Stroher et al., 2001).
   3. Optimal Temperature: 37 degrees celcius(Stroher et al., 2001).
   4. Optimal Humidity: humidity 95%(Stroher et al., 2001).
   5. Note: Diagnosis by viral cultivation and identification for the VHF-causing agents requires 3 to 10 days for most (longer for the hantaviruses); and, with the exception of dengue, specialized microbiologic containment is required for safe handling of these viruses(Website 7).

1. Outbreak Locations:
   1. Recorded cases of the disease are rare, and have appeared in only a few locations. While the 1967 outbreak occurred in Europe, the disease agent had arrived with imported monkeys from Uganda. No other case was recorded until 1975, when a traveler most likely exposed in Zimbabwe became ill in Johannesburg, South Africa and passed the virus to his traveling companion and a nurse. 1980 saw two other cases, one in Western Kenya not far from the Ugandan source of the monkeys implicated in the 1967 outbreak. The attending physician of this patient in Nairobi became the second case. Another human Marburg infection was recognized in 1987 when a young man who had traveled extensively in Kenya, including western Kenya, became ill and later died. In 1998, an outbreak occurred in Durba, Democratic Republic of the Congo. Cases were linked to individuals working in a gold mine. After the outbreak subsided, there were still some sporadic cases that occurred in the region(Website 8).
2. Transmission Information:
   1. From: Human(Website 8). , To: Human(Website 8). , With Destination:Human(Website 8).
      Mechanism: Just how the animal host first transmits Marburg virus to humans is unknown. However, as with some other viruses which cause viral hemorrhagic fever, humans who become ill with Marburg hemorrhagic fever may spread the virus to other people. This may happen in several ways. Persons who have handled infected monkeys and have come in direct contact with their fluids or cell cultures, have become infected. Spread of the virus between humans has occurred in a setting of close contact, often in a hospital. Droplets of body fluids, or direct contact with persons, equipment, or other objects contaminated with infectious blood or tissues are all highly suspect as sources of disease(Website 8).
   2. From: Primate(Website 8). , To: Human(Website 8). , With Destination:Human(Website 8).
      Mechanism: Persons who have handled infected monkeys and have come in direct contact with their fluids or cell cultures, have become infected(Website 8).
3. Environmental Reservoir:
   1. Currently no environmental reservoir information is available.
4. Intentional Releases:
   1. Intentional Release Information:
      1. Emergency Contact: If clinicians feel that VHF is a likely diagnosis, they should take two immediate steps: 1) isolate the patient, and 2) notify local and state health departments and CDC(MMWR, 1988). Report incidents to state health departments and the CDC (telephone {404} 639-1511; from 4:30 p.m. to 8 a.m., telephone {404} 639-2888). Information on investigating and managing patients with suspected viral hemorrhagic fever, collecting and shipping diagnostic specimens, and instituting control measures is available on request from the following persons at Centers for Disease Control (CDC) in Atlanta, Georgia; for all telephone numbers, dial 404-639 + extension: Epidemic Intelligence Service (EIS) Officer, Special Pathogens Branch, Division of Viral Diseases, Center for Infectious Diseases (ext. 1344); Chief, Special Pathogens Branch, Division of Viral Diseases, Center for Infectious Diseases: Joseph B. McCormick, M.D. (ext. 3308); Senior Medical Officer, Special Pathogens Branch, Division of Viral Diseases, Center for Infectious Diseases: Susan P. Fisher-Hoch, M.D. (ext. 3308); Director, Division of Viral Diseases, Center for Infectious Diseases (ext. 3574). After regular office hours and on weekends, the persons named above may be contacted through the CDC duty officer (ext. 2888)(MMWR, 1988).
      2. Delivery Mechanism: The VHF agents are all highly infectious via the aerosol route, and most are quite stable as respirable aerosols. This means that they satisfy at least one criterion for being weaponized, and some clearly have the potential to be biological warfare threats. Most of these agents replicate in cell culture to concentrations sufficiently high to produce a small terrorist weapon, one suitable for introducing lethal doses of virus into the air intake of an airplane or office building. Some replicate to even higher concentrations, with obvious potential ramifications. Since the VHF agents cause serious diseases with high morbidity and mortality, their existence as endemic disease threats and as potential biological warfare weapons suggests a formidable potential impact on unit readiness. Further, returning troops may well be carrying exotic viral diseases to which the civilian population is not immune, a major public health concern(Website 7).
      3. Containment: Patients with VHF syndrome generally have significant quantities of virus in their blood, and perhaps in other secretions as well (with the exceptions of dengue and classic hantaviral disease). Well-documented secondary infections among contacts and medical personnel not parenterally exposed have occurred. Thus, caution should be exercised in evaluating and treating patients with suspected VHF syndrome. Over-reaction on the part of medical personnel is inappropriate and detrimental to both patient and staff, but it is prudent to provide isolation measures as rigorous as feasible. At a minimum, these should include the following: stringent barrier nursing; mask, gown, glove, and needle precautions; hazard-labeling of specimens submitted to the clinical laboratory; restricted access to the patient; and autoclaving or liberal disinfection of contaminated materials, using hypochlorite or phenolic disinfectants. For more intensive care, however, increased precautions are advisable. Members of the patient care team should be limited to a small number of selected, trained individuals, and special care should be directed toward eliminating all parenteral exposures. Use of endoscopy, respirators, arterial catheters, routine blood sampling, and extensive laboratory analysis increase opportunities for aerosol dissemination of infectious blood and body fluids. For medical personnel, the wearing of flexible plastic hoods equipped with battery-powered blowers provides excellent protection of the mucous membranes and airways(Website 7).

1. Organism Detection Test:
   1. Electron Microscopy :
      1. Description: When the identity of a VHF agent is totally unknown, isolation in cell culture and direct visualization by electron microscopy, followed by immunological identification by immunohistochemical techniques is often successful(Website 7). Ebola, Marburg and Lassa viruses produced cytopathic effect in Vero cells, commencing at the 7th day post inoculation. In CV-1 cells, cytopathic effect was observed on the 5th day post inoculation for Ebola and on the 6th day post inoculation for Marburg and Lassa viruses. In both Vero and CV-1 cells, Ebola and Marburg virus particles were detected by electron microscopy three days post inoculation. Lassa particles were detected on the 4th and 6th days post inoculaton in CV-1 and Vero cells, respectively. In both cell lines, the three viruses were detected by EM before the appearance of cytopathic effect. Viral antigens of Ebola, Marburg and Lassa were detected by indirect immunofluorescence on the 3rd day post inoculation in Vero cells. In CV-1 cells, Marburg and Lassa antigens were detected on the 2nd day post inoculation, a day earlier than in Vero cells. While Ebola antigens were detected on the 3rd day post inoculation in both systems, fluorescent foci were much more pronounced in CV-1 than in Vero cells(Mekki and Van Der Groen, 1981). 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(Geisbert and Jahrling, 1995).
   2. Transmission electron microscopy :
      1. Description: 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(Geisbert and Jaax 1998).
   3. Immunofluorescence :
      1. Description: Ebola, Marburg and Lassa viruses produced cytopathic effect in Vero cells, commencing at the 7th day post inoculation. In CV-1 cells, cytopathic effect was observed on the 5th day post inoculation for Ebola and on the 6th day post inoculation for Marburg and Lassa viruses. In both Vero and CV-1 cells, Ebola and Marburg virus particles were detected by electron microscopy three days post inoculation. Lassa particles were detected on the 4th and 6th days post inoculaton in CV-1 and Vero cells, respectively. In both cell lines, the three viruses were detected by EM before the appearance of cytopathic effect. Viral antigens of Ebola, Marburg and Lassa were detected by indirect immunofluorescence on the 3rd day post inoculation in Vero cells. In CV-1 cells, Marburg and Lassa antigens were detected on the 2nd day post inoculation, a day earlier than in Vero cells. While Ebola antigens were detected on the 3rd day post inoculation in both systems, fluorescent foci were much more pronounced in CV-1 than in Vero cells(Mekki and Van Der Groen, 1981).
   4. Virus isolation in animals :
      1. Description: Innoculation of guinea pigs or monkeys, or sometimes mice or hamsters. Animals may become ill or require blind passage. This procedure is time consuming, expensive, and dangerous, but probably the most sensitive. BSL-4 laboratory is required(Peters et al., 1996).
2. Immunoassay Test:
   1. Hemagglutination :
      1. Description: Sera collected in May 1984 from 132 adult residents of Karamoja district, Uganda, were examined by haemagglutination inhibition tests for antibodies against selected arboviruses. A few individuals had antibodies against Crimean-Congo haemorrhagic fever, Lassa, Ebola and Marburg viruses, suggesting that these viruses all circulate in the area(Rodhain et al., 1989).
   2. Competitive and Two-Antibody ELISA :
      1. Description: Comparative studies of two variants of the enzyme-linked immunosorbent assay (ELISA) were carried out to determine the sensitivity of the detection of Marburg virus antigens in Vero cells. Both competitive and two-antibody ELISA variants detected as little as 5 ng of Marburg virus antigen. The Vero cell monolayer was found to produce 5-50 ng/0.05 ml of the virus-specific proteins at 6 to 8 days postinfection(Vladyko et al., 1991). Detection of either Ebola or Marburg virus antigen by antigen-capture enzyme-linked immunosorbent assay (ELISA) has often been the most rapid means of diagnosing infections. The assay time is approximately 3 to 4 hours, and detection of antigen in the blood or serum of either nonhuman primates or patients in the acute course of the disease was very successful when compared to virus isolation or to RT-PCR for Ebola viruses. This assay was also useful in detection of virus antigen in frozen tissues(Sanchez et al., 2001).
   3. IgM-capture antibody ELISA :
      1. Description: Saijo et al. (2001) developed enzyme-linked immunosorbent assays (ELISA) using recombinant filovirus nucleoproteins (NPs) to detect immunoglobulin G (IgG) antibodies to Ebola (EBO) and Marburg (MBG). They demonstrated that the ELISA had high sensitivity and specificity for detection of EBO and MBG antibodies(Saijo et al., 2001). All the MBG patients' sera tested MBG antibody positive. Furthermore, the specificity of the IgG ELISA was 100%. These results suggest that the ELISA may be useful not only for diagnosis of surviving MBG-infected patients but also for epidemiological field studies(Saijo et al., 2001). IgM-capture antibody ELISA proved useful in the diagnosis of recent infections in surviving patients. The IgG response of the patients was somewhat delayed and realistically could be expected only in the early convalescent sera of those patients who were destined to recover from their infections. The IgM antibodies detected by this test have been found to persist for only 2 to 3 months in macaques that were experimentally infected and a similar relatively short period in surviving humans(Sanchez et al., 2001).
   4. ELISA :
      1. Description: In the present serological study 120 monkey sera from different species originating from the Philippines, China, Uganda and undetermined sources and several groups of human sera comprising a total of 1288 specimens from people living in Germany were examined for the presence of antibodies directed against filoviruses (Marburg virus, strain Musoke/Ebola virus, subtype Zaire, strain Mayinga/Reston virus). Sera were screened using a filovirus-specific enzyme-linked immunosorbent assay (ELISA). ELISA-positive sera were then confirmed by the indirect immunofluorescence technique, Western blot technique, and a blocking assay, and declared positive when at least one confirmation test was reactive(Becker et al., 1992).
   5. Indirect Fluorescent Antibody (IFA) :
      1. Description: IFA is widely used. It is adaptable to field situations, if fluorescnt microscope is available. Until recently, it was the most reliable test available(Peters et al., 1996).
   6. Enzyme immunoassay :
      1. Description: An investigation was conducted between 1994 and 1997 in forested areas of the Central African Republic (CAR) to determine the seroprevalence of IgG antibodies against several haemorrhagic fever viruses present in the region. Sera were obtained from 1762 individuals in two groups (Pygmy and Bantu locuted populations) living in 4 forested areas in the south of the country. Sera were tested for IgG antibodies against Ebola, Marburg, Rift Valley fever (RVF), Yellow fever (YF) and Hantaviruses by enzyme immunoassay (EIA), and against Lassa virus by immunofluorescent assay. The prevalence of IgG antibodies was 5.9% for Ebola, 2% for Marburg, 6.9% for RVF, 6.5% for YF, 2% for Hantaan. No antibodies were detected against Lassa, Seoul, Puumala and Thottapalayam viruses. No IgM antibodies were detected against RVF and YF viruses. The distribution of antibodies appears to be related to tropical rain forest areas(Nakounne et al., 2000).
3. Nucleic Acid Detection Test: