PHIDIAS: Pathogen-Host Interaction Data Integration and Analysis System

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

Taxonomy Information

Species:
1. Ebola virus (Website 3):
  1. Common Name: Ebola
  2. GenBank Taxonomy No.: 11268
  3. Description: Ebola virus is a nonsegmented RNA virus, which, together with Marburg virus, makes up the filovirus family(Nichol et al., 2000). Members of the family filoviridae are negative-strand ssRNA viruses(Website 3). In contrast to most RNA viruses, EBOV is characterized by high genetic stability, which may be due to four main factors: low error rate of RNA polymerase, slow replication in the natural host, small number of natural hosts and weak immunological pressure(Leroy et al., 2002).
  4. Variant(s):
    - Zaire Ebola virus (Website 5):
      - Common Name: Zaire Ebola virus
      - GenBank Taxonomy No.: 186538
      - Parents: Ebola virus
      - Description: Ebola first occurred in Yambuku and surrounding area. Disease was spread by close personal contact and by use of contaminated needles and syringes in hospitals/clinics. This outbreak was the first recognition of the disease(Website 25). Ebola virus is subdivided into four subtypes, namely Zaire, Sudan, Cote-d'Ivoire, and Reston(Leroy et al., 2002).
    - Ebola virus strain Eckron-76 (Website 6):
      - Common Name: Ebola virus strain Eckron-76
      - GenBank Taxonomy No.: 129000
      - Parents: Zaire Ebola virus
      - Description: The EBOV strain Eckron, subtype Zaire, as EBOV strain Mayinga, was isolated from a case of the 1976 outbreak of Ebola hemorrhagic fever in Zaire and represents a wild-type strain(Volchkov et al., 1998).
    - Ebola virus strain Gabon-94 (Website 7):
      - Common Name: Ebola virus strain Gabon-94
      - GenBank Taxonomy No.: 128947
      - Parents: Zaire Ebola virus
      - Description: Gabon has recently been struck three times by Ebola hemorrhagic fever(Volchkov et al., 1997). This epidemic occurred in Mekouka and other gold-mining camps deep in the rain forest. Initially thought to be yellow fever; it was identified as Ebola hemorrhagic fever in 1995(Website 25).
    - Ebola virus strain Zaire Mayinga (Website 8):
      - Common Name: Ebola virus strain Zaire Mayinga
      - GenBank Taxonomy No.: 128952
      - Parents: Zaire Ebola virus
      - Description: The EBOV strain Eckron, subtype Zaire, as EBOV strain Mayinga, was isolated from a case of the 1976 outbreak of Ebola hemorrhagic fever in Zaire and represents a wild-type strain(Volchkov et al., 1998).
    - Ebola virus strain Zaire-95 (Website 9):
      - Common Name: Ebola virus strain Zaire-95
      - GenBank Taxonomy No.: 128951
      - Parents: Zaire Ebola virus
      - Description: Ebola hemorrhagic fever (EHF) patients treated at Kikwit General Hospital during the 1995 outbreak were tested for viral antigen, IgG and IgM antibody, and infectious virus. Virus was also isolated from patients during the course of their febrile illness(Ksiazek et al., 1999).
    - Sudan Ebola virus (Website 10):
      - Common Name: Sudan Ebola virus
      - GenBank Taxonomy No.: 186540
      - Parents: Ebola virus
      - Description: The first outbreak in Sudan occurred in Nzara, Maridi and the surrounding area. Disease was spread mainly through close personal contact within hospitals(Website 25). Many medical care personnel were infected. Ebola virus is subdivided into four subtypes, namely Zaire, Sudan, Cote-d'Ivoire, and Reston(Leroy et al., 2002).
    - Ebola virus strain Sudan Boniface (Website 11):
      - Common Name: Ebola virus strain Sudan Boniface
      - GenBank Taxonomy No.: 128948
      - Parents: Sudan Ebola virus
      - Description: Ebola viruses-Sudan (S/Sudan/Maridi/1976/VCP2D11 and S/Sudan/Nzara/1979/015176) are also known a Boniface and Maleo, respectively(Sanchez et al., 1996). A new viral disease (Maridi haemorrhagic fever) occurred in the South Sudan in 1976. It was obviously identical with an epidemic which occurred at the same time in Zaire(Knobloch et al., 1977).
    - Ebola virus strain Sudan Maleo-79 (Website 12):
      - Common Name: Ebola virus strain Sudan Maleo-79
      - GenBank Taxonomy No.: 128949
      - Parents: Sudan Ebola virus
      - Description: Occurred in Nzara. Recurrent outbreak at the same site as the 1976 Sudan epidemic(Website 25).
    - Cote d'Ivoire Ebola virus (Website 15):
      - Common Name: Cote d'Ivoire Ebola virus
      - GenBank Taxonomy No.: 186541
      - Parents: Ebola virus
      - Description: Ebola virus is subdivided into four subtypes, namely Zaire, Sudan, Cote-d'Ivoire, and Reston (Leroy et al., 2002)(Leroy et al., 2002). In this instance a scientist became ill after conducting an autopsy on a wild chimpanzee in the Tai Forest. The patient was treated in Switzerland(Website 25).
    - Ebola virus strain Ivory coast-94 (Website 16):
      - Common Name: Ebola virus strain Ivory coast-94
      - GenBank Taxonomy No.: 128999
      - Parents: Cote d'Ivoire Ebola virus
      - Description: A new strain of Ebola virus was isolated from a non-fatal human case infected during the autopsy of a wild chimpanzee in the Cote-d'Ivoire. The wild troop to which this animal belonged had been decimated by outbreaks of haemorrhagic syndromes. This is the first time that a human infection has been connected to naturally-infected monkeys in Africa(Le Guenno et al., 1995).
    - Reston Ebola virus (Website 13):
      - Common Name: Reston Ebola virus
      - GenBank Taxonomy No.: 186539
      - Parents: Ebola virus
      - Description: Ebola virus is subdivided into four subtypes, namely Zaire, Sudan, Cote-d'Ivoire, and Reston(Leroy et al., 2002). Ebola-Reston virus was introduced into quarantine facilities in Virginia, Texas, and Pennsylvania by monkeys imported from the Philippines. Four humans developed antibodies to Ebola-Reston virus but did not become ill(Leroy et al., 2002, Website 25).
    - Ebola virus strain Reston Siena/Philippine-92 (Website 14):
      - Common Name: Ebola virus strain Reston Siena/Philippine-92
      - GenBank Taxonomy No.: 129004
      - Parents: Reston Ebola virus
      - Description: Ebola-Reston virus was introduced into quarantine facilities in Sienna by monkeys imported from the same export facility in the Philippines that was involved in the episodes in the United States. No humans were infected(Website 25).

Lifecycle Information

Ebola 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 (Beer and Kurth, 1999).
    - Picture(s):
      - Ebola virus from the first passage in Vero cells (Website 30)
        
        Description: Ebola virus, diagnostic specimen from the first passage in Vero cells of a specimen from a human patientthis image is from the first isolation and visualization of Ebola virus, 1976. This image has been "borrowed" so often for various public uses that many people think that all Ebola virions look just like thisindeed, in the film Outbreak every virion seen looked just like this. In fact, Ebola virions are extremely varied in appearance they are flexible filaments with a consistent diameter of 80 nm (nanometers), but they vary greatly in length (although their genome length is constant) and degree of twisting. Negatively stained virions. Magnification: approximately x60,000. Micrograph from F. A. Murphy, School of Veterinary Medicine, University of California, Davis.

Genome Summary

Genome of Zaire Ebola virus
1. Description: The first incidence of Ebola Zaire occurred in Yambuku and surrounding area. Disease was spread by close personal contact and by use of contaminated needles and syringes in hospitals/clinics. This outbreak was the first recognition of the disease. 318 people were infected and 88% of them died(Website 25).
2. Chromosome(Website 18, Website 19, Website 20, Website 21)
  1. GenBank Accession Number: AY142960 AF499101 NC_002549 AF086833
  2. Size: 18959 base pairs(Website 18, Website 19, Website 20, Website 21).
  3. Gene Count: 7 genes(Website 18, Website 19, Website 20, Website 21).
  4. Description: The genomes of filoviruses display linear arranged genes on a single negative-stranded RNA molecule that encode the seven structural proteins in the order nucleoprotein, virion structural protein (VP) 35, VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L)(Volchkov et al., 1998).
Genome of Reston Ebola virus
1. Description: The Reston species of EBOV is unique in that it originated in Asia, while the other filoviruses have their origins in Africa. EBOV Reston emerged in 1989/90 as the causative agent of an epizootic among a group of cynomolgus monkeys (Macaca fascicularis) imported from the Philippines into the United States. Subsequently, at least two more introductions of EBOV Reston have occurred in the United States and Italy. Epidemiological investigations in the Philippines have documented active virus transmission in the primate export facility which was the source of all the shipments of infected monkeys. Despite pathogenicity for non-human primates, EBOV Reston has never been associated with any notable disease in man. However, serological investigations documented at least eight seroconversions among exposed animal handlers, which suggests that EBOV Reston infections in humans are infrequent and lead to either asymptomatic or subclinical courses(Groseth et al., 2002).
2. Chromosome(Website 22, Website 23, Website 24)
  1. GenBank Accession Number: AB050936 AF522874 NC_004161
  2. Size: 18890 or 18891 base pairs(Website 22, Website 23, Website 24).
  3. Gene Count: 7 genes(Website 22, Website 23, Website 24).
  4. Description: The genomes of filoviruses display linear arranged genes on a single negative-stranded RNA molecule that encode the seven structural proteins in the order nucleoprotein, virion structural protein (VP) 35, VP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L)(Volchkov et al., 1998).

Biosafety Information

Biosafety information for Ebola virus
1. Level: Virus isolation must only be attempted in Biosafety Level 4 facilities, such as are available at CDC.
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).

Culturing Information

General Cell Culture Information for Ebola Virus :
1. Description: Virus isolation in cell culture. Acute sera or postmortem tissues are usually positive. Virus growth is detected by cytophatic 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).
In Vitro Culture System :
1. Description: To study the host response to and identify potential diagnostic markers for Ebola virus infections, an in vitro culture system involving nonhuman primate alveolar macrophages was developed and characterized(Gibb et al., 2002). Data from this study indicate clearly that nonhuman primate alveolar macrophages are susceptible to Ebola virus infection in vitro and are capable of replicating the virus to a high titer within 24 h. These data are particularly significant, as alveolar macrophages are the first line of phagocytic defense against inhaled pathogens and are known to be important in orchestrating the host immune response (i.e., cytokine production and regulation of MHC molecule expression) to infectious agents that reach the alveoli . In this system, Ebola virus replication appeared to proceed efficiently. Within 24 h of initial infection, viral replication led to irreversible cell injury and severe cytolysis(Gibb et al., 2002).
2. Medium: Viral stock was diluted serially in minimal essential medium with Earle salts and nonessential amino acids, adsorbed to confluent Vero E6 cells in 12-well dishes, incubated for 1 h at 37 degrees celcius, and covered with an agarose overlay(Gibb et al., 2002).
3. Optimal Temperature: 37 C(Gibb et al., 2002).
4. 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 28).
Vero E6 Cell Culture :
1. Description: In this study they used the Musoke strain of Marbug 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 C(Stroher et al., 2001).
4. Optimal 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 28).

Epidemiology Information:

Outbreak Locations:
1. Ebola viruses are taxonomically related to Marburg viruses; they were first recognized in association with explosive outbreaks that occurred almost simultaneously in 1976 in small communities in Zaire and Sudan(Website 28). Sporadic cases occurred subsequently. In 1989, a third strain of Ebola virus appeared in Reston, Virginia, in association with an outbreak of VHF among cynomolgus monkeys imported to the United States from the Philippines(Website 28). Recently, small outbreaks involving new strains of Ebola virus occurred in human populations in Cote d'Ivorie in 1994 and Gabon in 1995; a larger outbreak involving the Ebola-Zaire strain involved more than 300 people, with 75% mortality, in Zaire in 1995(Website 28). On the basis of serological evidence, the geographic distribution of EBO viruses may include other parts of Asia and Madagascar(Monath, 1999).
Transmission Information:
1. From: Human(Website 27). , To: Human(Website 27). , With Destination:Human(Website 27). (Website 27)
  Mechanism: The source of infection or mode of transmission of Ebola virus to human index cases of Ebola fever has not been established. Field observations in outbreaks of Ebola fever indicate that secondary transmission of Ebola virus is linked to improper needle hygiene, direct contact with infected tissue or fluid samples, and close contact with infected patients. While it is presumed that the virus infects through either breaks in the skin or contact with mucous membranes, the only two routes of exposure that have been experimentally validated are parenteral inoculation and aerosol inhalation(Jaax et al., 1996). While all Ebola virus species have displayed the ability to be spread through airborne particles (aerosols) under research conditions, this type of spread has not been documented among humans in a real-world setting, such as a hospital or household(Website 27).
2. From: Primates(Formenty et al., 1999). , To: Human(Formenty et al., 1999). , With Destination:Human(Formenty et al., 1999). (Formenty et al., 1999)
  Mechanism: The chimpanzee organs from the implicated necropsy were shown by immunohistochemistry to be infected with Ebola. During necropsy, all 3 researchers had worn gloves but not masks or gowns. No wounds or punctures had been noted. Two researchers had worn latex examination gloves, but the case-patient had worn household gloves. Therefore, it is highly probable that she became contaminated during the necropsy by direct contact with chimpanzee blood on her hand or by projection of droplets onto her face(Formenty et al., 1999). Human infections resulting from exposure to wild, infected chimpanzees butchered for meat (in Gabon, 1996) or autopsied because of illness (in Cote d'Ivorie, 1994) have provided the most direct clues to the source of EBO virus in nature(Monath, 1999).
3. From: Bats(Monath, 1999). , To: Human(Monath, 1999). , With Destination:Human(Monath, 1999). (Monath, 1999)
  Mechanism: Evidence implicating bats is circumstantial and includes the transcontinental geographic distribution of EBO virus subtypes, the association between infection and potential roosting sites for bats in caves (Marburg virus; Kitum cave) or in man-made structures (EBO-S virus; cotton factory, Nzara, Sudan), and the association between bats a rhabdoviruses and paramyxoviruses(Monath, 1999).
Environmental Reservoir:
1. Currently no environmental reservoir information is available.
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, 1998). 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 28).
  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 28).

Diagnostic Tests Information

Organism Detection Test:
1. Electron microscopy :
  1. Time to Perform: 2-to-7-days
  2. 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 28). 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).
2. Indirect Immunofluorescence :
  1. Time to Perform: 2-to-7-days
  2. 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).
3. Immunofluorescence Microscopy :
  1. Description: A novel recombinant baculovirus which expresses Ebola virus (EBO) nucleoprotein (NP) under the control of the cytomegalovirus immediate-early promoter was constructed. HeLa cells abortively infected with the baculovirus expressed EBO NP, and this was used as an immunofluorescent (IF) antigen to detect EBO immunoglobulin G (IgG) antibody. This IF method has high efficacy in detecting EBO IgG antibody in clinical specimens, indicating its usefulness in the diagnosis of EBO infections and seroepidemiological studies(Saijo et al., 2001). In previous reports on the seroepidemiology of EBO, inactivated EBO-infected cells were used as antigens for indirect IF methods. Our IF method may offer an attractive alternative to the use of live EBO-infected cells, which should be handled in a biosafety level 4 laboratory(Saijo et al., 2001).
  2. False Positive: Among the 48 sera from subjects without a history of EBO infections, one showed positive staining at a dilution of 1:100. We could not rule out the possibility that this serum had IgG antibodies specific to EBO. Even if the positive reaction by 1 of the 48 control sera was nonspecific, the IF system using rEBO NP-expressing HeLa cells has high efficacy in detecting EBO-specific IgG, with 100% sensitivity and 98% specificity. The number of serum samples used for the evaluation of this IF system was small; these results indicate the usefulness of this IF method for detection of EBO-specific IgG antibody(Saijo et al., 2001).
4. Virus isolation in animals (Peters et al., 1996):
  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 required(Peters et al., 1996).
5. Indirect immunoelectron microscopy :
  1. Time to Perform: 1-hour-to-1-day
  2. Description: 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(Geisbert et al., 1991).
Immunoassay Test:
1. Haemagglutination :
  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. ELISA using Antibody against Nucleoprotein :
  1. Description: 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(Niikura et al., 2001). One of the advantages of our new ELISA system is that the highest level of security containment is not required, since no live virus except that in the clinical specimens is involved. This means that an ordinarily equipped laboratory can reproduce the system as long as the secondary polyclonal antibody, which can also be prepared by use of noninfectious recombinant protein, is available. The disadvantage is that it uses only one MAb that recognizes at least three of four subtypes of Ebola virus. Although Ebola virus is genetically stable and so far only four subtypes have been reported, there is no guarantee that this MAb cross-reacts with all the minor variants that might appear in the future(Niikura et al., 2001). 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. EIA :
  1. Description: 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 less than 90 days. The IgG response was less rapid; however, it persisted for more than 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(Ksiazek et al., 1999). 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 using Antibody against Glycoprotein :
  1. Description: Monoclonal antibodies to EBOV were produced from mice immunized with inactivated EBOV species Zaire. Antibodies directed against the viral glycoprotein GP were characterized by ELISA, Western blot and immunofluorescence analyses. An antigen capture ELISA was established, which is specific for EBOV-Zaire and shows a sensitivity of approximately 10(3) plaque-forming units/ml. Since the ELISA is able to detect even SDS-inactivated EBOV in spiked human sera, it could complement the existing diagnostic tools in the field and in routine laboratories where high containment facilities are not available. The sensitivity of the assay corresponded to approximately 10(3) pfu/ml serum(Lucht et al., 2003).
5. ELISA using Antibody against Reston Nucleoprotein :
  1. Description: Antigen capture ELISAs was developed using two novel MAbs, Res2-6C8 and Res2-1D8, specific to the NP of EBO-R. Res2-6C8 and Res2-1D8 recognized epitopes consisting of 4 and 8 amino acid residues, respectively, near the C-terminal region of the EBO-R NP. The antigen capture ELISAs using these two MAbs detected the EBO-R NP in the tissues from EBO-R-infected cynomolgus macaques. The antigen capture ELISAs using Res2-6C8 and Res2-1D8 are useful for the rapid detection of the NP in EBO-R-infected cynomolgus macaques (Ikegami et al., 2003). The ELISA prepared with 3-3D detected His-EBO-R-NP upto the dilution of 1:32,000(Ikegami et al., 2003).
6. ELISA using Antibody against VP40 :
  1. Description: An antigen capture enzyme-linked immunosorbent assay was established, which detects VP40 of all known species of EBOV. This assay could detect viral material in spiked human serum that has been sodium dodecylsulfate-inactivated. The established enzyme-linked immunosorbent assay therefore has the ability to become a very useful tool for obtaining an accurate diagnosis in the field, limiting the risk of laboratory infections. The sensitivity of the assay corresponded to 10(3) to 10(4) pfu/ml serum(Lucht et al., 2003B).
7. Western Blotting using ABs against Nucleoprotein and VP40 :
  1. Description: Purified Ebola virus from tissue culture medium was loaded on 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane at 55 V for 3 h at 4 degrees celcius. Protein immunoblotting analysis was done with serum samples diluted in a ratio of 1:500, and a secondary antibody (goat antibody to human IgG peroxidase conjugate) in a dilution of 1:30000. All incubations were done for 1 h at room temperature (Leroy et al., 2000). Western-blot analysis showed that IgG responses were directed to nucleoprotein and viral protein of 40 kDa(Leroy et al, 2000B).
8. Enzyme immunoassay :
  1. Description: Enzyme immunoassay (EIA) test systems for the detection of antigens and antibodies to Ebola virus were developed and tried. The test system for the detection of Ebola virus antigens based on direct solid-phase EIA detects viral antigens in culture fluid of infected Vero cells, in the blood sera, and in homogenates of infected tissues. Use of this test system allows detection of at least 10 ng of viral proteins or 5.0 x 10(3) to 1.0 x 10(4) PFU/ml in infectious material. The test system is prepared on the basis of protein A - horseradish peroxidase conjugate. It is universal for the testing of animal and human sera and is characterized by high resolution and reproducibility of results. It allows detection of antibodies to Ebola virus starting from days 8-9 of infection. A higher sensitivity of direct solid-phase EIA in comparison with complement fixation or indirect immunofluorescence tests is demonstrated(Merzlikin et al., 1995).
9. 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).
10. Immunofluorescence focus assay :
  1. Time to Perform: 1-to-2-days
  2. Description: A 48-h indirect immunofluorescence focus assay for the quantitation of Ebola virus was developed, utilizing HeLa-229 cell monolayers. The dose dependency and the sensitivity of this assay as compared with conventional assays are reported. This indirect immunofluorescence focus assay can be used as a rapid, quantitative test for the detection of Ebola virus(Truant et al., 1983).
Nucleic Acid Detection Test:


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