The Filoviruses (Latin – Filo – Threadlike) are a relatively new family of viruses, consisting of only 2 known serotypes – Ebola virus and
The Filoviridae family has been known to science since 1967 when an outbreak of hemorrhagic fever infected 31 lab workers in
The infrequency of
In 1976 a new threat emerged in
This was the emergence of Ebola Hemorrhagic fever. So called after the river in the Democratic
The incubation period varies from between 2 & 16 days, depending on the strain.
Filoviruses announce their presence in the form of severe haemorrhagic fever, accompanied with chills, myalgia, abdominal pain, pharyngeal and vasodilation (DM Sander 1993).
Death usually follows shock from blood and fluid loss
No effective vaccine exists at present. Interferon has been used as an antiviral treatment, although significant resistance persists (DM Sander 1993).
Treatment is mainly supportive, ie – fluid maintenance, painkillers and sedatives. There have been reports of steroids reducing the worse of the symptoms of Ebola/Marburg virus infection.
The vast majority of Ebola related outbreaks occur across the humid Afro tropics – The Democratic Republic of Congo, Gabon, Zaire, Uganda, Kenya, Sudan and Tanzania, (Peterson)with the exception of E. Sudan which may occur in tandem with Marburg virus, which is restricted to the more drier, isolated areas of Botswana, Zimbabwe, Ethiopia and Sudan
Ebola Reston - Ebola Reston was first discovered in
Marburg & Ebola are (or would appear to be) indigenous to the African continent. Ebola has only been isolated from individuals in or from
Ebola Reston has been detected in captive non-human primates in
Serological testing has indicated that Filoviruses are endemic in many central African countries. Recent sero-surveys indicate that Filoviruses may be endemic in many other countries including
Intimate contact is the main cause of infection in person - person transmission. Transmission of Filoviruses is (thankfully) relatively inefficient, as secondary infection rates seldom exceed 10%. Nosocomial transmission (infection from a health care environment) from contaminated needles and syringes poses a serious problem, as was documented in the 1976 & 1995 outbreaks in
Transmission from droplets and airborne particles has been observed experimentally in infected monkeys.
In September 1983,
Aerosols and droplets are not effective in a natural environment, although possible, as 1 droplet of infected blood may contain over 100 million virus particles (
Tai national park
Gabon & DRC
October 2004 saw the start of an outbreak of
The last known Ebola virus outbreak was in the DRC in February 18th 2003. The out break peaked at 187 deaths from 202 confirmed cases, the classic mortality rate of 92% for Ebola
Filoviruses may enter the host via skin lesions and mucous membranes, and so access the blood directly or via the lymphatic system
Filovirus infections tend to follow a specific order, starting with blood or lymph fluid (interstitium) and the parenchyma (functional elements of the tissue or organ etc). Pathogenic effects take place in the blood stream. Mononuclear phagocytic cells, especially macrophages appear to be the primary targets of Filoviruses. Virus production in mononuclear phagocytic cells causes high viremia (high virus levels in the blood stream) during early stages of infection (Roitt 1991).
Filovirus infection causes the activation of monocytes, which in turn causes clump formation and the release of mediators. Cytokines are also produced (TNF & IL-1) along with pro-inflammatory mediators, which are known to increase the expression of various cell adhesion molecules on the surface of endothelial cells, (ICAM-1, VCAM-1, E-selectin & P-selectin) which have been found to be responsible for diapedesis (The passage of blood cells through vessel walls) of the Leukocytes (American Society of Microbiology 2002).
Extravasation of infected monocytes is thought to be the method by which the virus spreads from the blood stream to the organ tissues during the secondary infection, possibly explaining the pan-tropism often documented. This method of infection is also exercised in HIV and the visna virus (ASM 2002).
Monocyte infection leads to the activation and release of cytokines and mediators, (Figure 4 – ASM 2002) cytokines cause the up regulation of cell adhesion molecules, increased procoagulation activity and the inevitable para – endothelial permeability (ASM 2002). Infected monocytes participate in the spread of the virus though out the host.
The clumping of monocytes is essential in the pathogenesis of Filoviruses, as monocyte clumps in the vessels influence the ‘flow’ of the blood stream, with the potential to cause thrombus formation This has been observed in infected patients. High monocyte and macrophage levels induce the release of numerous cytokines, known to contribute to shock.
The supernatants of virally infected macrophages increase para-endothelial permeability in endothelial macrophages. This effect is mainly driven by TNF (Tumor necrosis factor).
Para-endothelial permeability is a major contributor of shock. Shock is the number 1 cause of death in Filovirus infection (Feldmann 1997).
Mononuclear phagocytic cells play an important role in pathogenesis by mediating virus spread from extravasation and the triggering of inter – related pathological host responses, which may lead to compromised immunity, impairment of homeostasis and the function of the endothelium. The neutralization of antibodies directed against inflammatory cytokines and chemokines may be the key to helping the host recover from the early events in infection by delaying viral progression sufficiently in order to allow the host time to mount an appropriate immune response.
Often referred to as the red death, the symptoms of Filovirus infections are severe. The start of the prodromal period is marked by the onset of acute headaches after approximately 4 days from initial infection (although symptoms have been known to appear as early as 2 days), often accompanied by general aches and pains (Feldmann 1997). By the 2nd day the patient will have fever, progressing to diarrhea, abdominal pain, nausea, vomiting and a sore throat and anorexia. By day 7 the patient will have a maculopapular (small raised spots) rash and develop thrombocytopenia and have haemorrhagic manifestations, most noticeably in the gastrointestinal tract and lungs, but it can and does occur from any orifice, mucus membrane or skin (Leicester 2000). By day 12 the skin starts to peel from the rash. Mental disturbances have also been documented by this stage due to encephalitis.
Ebola in particular causes lesions in every organ, most noticeably on the liver and spleen, both showing signs of enlargement and necrosis, Ebola infection has been said to turn every organ into a ‘jelly like’ consistency. Death normally occurs by day 13 (often far sooner) from massive hemorrhage due to diapedesis and disseminated intravascular coagulation (DIC). End stage Ebola infection is indicated by haemorrhage from the eyes, nose, ears, anus, urethra and mouth. As stated earlier, haemorrhage occurs from every possible orifice
‘The haemorrhagic and connective tissue complications may be due to the fact that the vp40 protein is antigenically related to human cll matrix proteins (abdominal aortic aneurism protein MFAP-4), leading to autoimmune attack’ (Leicester 2000).
Filoviruses are classed as biological level 4 agents, or ‘hot agents’, due to their high mortality rate, person to person transmission, potential for airborne infection and the lack of any effective treatment or vaccines. It is for these reasons that every conceivable precaution must be undertaken when dealing with them.
There is no cross-reactivity between the 2 types, although sub-types of Ebola share similar epitopes.
Comparisons of Ebola and
Electron micrograph x10,000(CDC 2001)
Ebola Zaire - x10,000 (CDC(2001)
When viewed from the electron microscope, Ebola appears noticeably longer and rather ‘threadlike’, with a distinct tangle at one end The Ebola virus is often found in such numbers within the cell (especially liver) that it resembles a mass of worms.
Viral genome -
Filoviruses consist of 7 proteins – NP, VP35, VP40, GP, VP30, VP24 & L. The length of the genome ranges from 18,000 – 19,100 bases (Ebola - 18,900.
VP35 & VP30 – Components of the ribonucleoprotein. VP30 is the phosphoprotein, VP35 is not phosphorylated. VP30 plays a role as a cofactor for transcriptase and replicase.
VP40 & VP24 – Membrane associated proteins, they are of a hydrophobic nature and found in abundance in virus particles. VP40 &VP24 are matrix proteins.
L – Large protein. The L protein is an RNA dependant RNA polymerase, containing many hydrophobic amino acid residues (Feldmann 1997).
GP – The glycoproteins are directed into the E.R by an N terminal hydrophobic area and cleaved by signal peptidases and anchored by the C terminal hydrophobic domain in the membrane. The glycoprotein is an integral protein and the only known glycosylated virion protein which contains both N & O linked oligosaccharides (Leicester 2000). The production of the glycoprotein involves ribosomal frame shifting, glycosylation & proteolytic cleavage of the precursor into GP 1 /GP 2 (Leicester 2000).
Filovirus glycoprotein sequence –
5’MKTTCLFISLILIQGIKTLPILEIASNNQPQNVDSVCSGTLQKT EDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSG KSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGRVF TEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSNNGTQTNDTGCFGALQEY NSTKNQTCAPSKIPSPLPTARPEIKPTSTPTDATTLNTTDPNNDDEDLITSGSGSGEQ EPYTTSDAVTKQGLSSTMPPTPSPQPSTPQQEGNNTDHSQGTVTEPNKTNTTAQPSMP PHNTTAISTNNTSKNNFSTLSVSLQNTTNYDTQSTATENEQTSAPSKTTLPPTGNLTT AKSTNNTKGPTTTAPNMTNGHLTSPSPTPNPTTQHLVYFRKKRSILWREGDMFPFLDG LINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGEN ENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQT AKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSRNISEQI DQIKKDEQKEGTGWGLGGKWWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG 3’
The glycoprotein has been observed as 2 distinct forms – GP1 & GP2. GP1 has a stop codon in the middle, preventing synthesis of the full protein. Infected cells have been found to be edited, the edited GP contains an extra adenosine at points 1019 – 1026 (Leicester 2000), this causes a frame shift mutation which allows full transcription of the protein. The larger protein is believed to be membrane associated and the smaller is secreted.
It is believed that this is to protect the virus from the immune system by acting as a decoy. The viral glycoproteins also act as immunosuppressive s by binding to and interfering with neutrophils, further diminishing the host’s response (WHO 2003).
This may be an area for developing future vaccines. ie- raising antibodies against the glycoprotein.
The viral glycoprotein is thought to be the major antigenic molecule.
The nonsegmented negative-stranded RNA genome is transcribed into subgenomic RNA’s and polyadenylated at their 3' and capped at their 5' ends. Replication runs from a positive strand anti-genome which acts as a template for synthesis of the negative stranded genome. Transcription and replication occurs in the cytoplasm and are controlled by the polymerase L protein along with cofactors (Feldmann 1997).
Entry may be controlled by GP’s, as these are the only viral surface proteins, although studies on infection of hepatocytes have identified the asialoglycoprotein receptor as a possible influence of cell entry, however, this receptor is not expressed on many virus-susceptible cells. It is not known if the next step in virus entry involves a fusion process at the plasma membrane or fusion following endocytosis of virus particles.(As is the main method of entry for many viruses). The uncoating process has not yet been studied for Filoviruses (Leicester 2000). Filovirus transcription and replication take place in the cytoplasm of infected cells ‘Transcription starts with a positive leader sequence, and the genomes are transcribed into monocistronic subgenomic RNA (mRNA) species complementary to viral genomic RNA’ (Feldmann 1997). The 3' ends of the transcripts carry a poly A tail generated by an unknown mechanism of the viral polymerase at a run of uridine residues located in the transcription & termination signals. The 5' ends of the transcripts are capped, and the sequences form hairpin structures, which may play a role in transcript stability and ribosome binding. (Feldmann 1997) The surrounding semi conserved sequences mediate the initiation of transcription and termination. The role of gene overlaps are unknown. The polymerase may terminate transcription at the overlap and initiate transcription of the downstream gene. ‘The switch mechanism between transcription and replication has not been studied.’ (Feldmann 1997). New virions bud from the plasma membrane.
Histopathology in diagnosis
Hepatocytes undergoing cell lysis. Viral particles can be observed spilling from the cells. Nuclei are darkened and necrotized. Formalin fixed tissue, observed from E.M. x12,500 (Murphy 2001)
High virus titres can be obtained from the liver, spleen, lymph nodes and the lungs once first symptoms appear. (As observed in African green monkeys)All these organs, especially the liver, are totally necrotized, with very little inflammatory response. Massive G.I haemorrhage occurs in the majority of cases. Thrombocytopenia and aggregation disorders of the platelets are the result of agonists such as ADP and collagen (Saunders 2004). High AST and ALT levels indicate severe liver damage. Extensive virus replication has been found in macrophages (tissue) and fibroblasts of all major organs (again especially the liver.), but less frequently in endothelial cells, adrenal cortoid cells and renal tubular epithelium. Connective tissue around the face deteriorates, giving the patient an expressionless appearance (
Liver samples from Ebola and
Often exceptionally large inter cytoplasmic eosinophilic inclusion bodies can be observed in the hepatocytes. The staining of the inclusion bodies are useful in diagnosis, however, this may be difficult due to the presence of councilman-like bodies (viral inclusions) in areas of necrosis (As one often resembles the other) In incidents where virus isolation and immuno-florescence methods are not possible, Ebola/Marburg infection may be determined by the examination of thin-section electron microscopy of formalin fixed tissue samples.
Hepatocellular inclusion bodies can be seen to be made up of a mass of tubules, identical to that of the viral particles, and indistinguishable from those found in Vero cell cultures, monkeys and guinea pigs infected with a Filovirus (Murphy 2001).
High numbers of virus particles (saturation point in some cases) can be found in all extracellular spaces, sinusoids, spaces of disease and areas of infection. The kidney shows varying states of necrosis and calcification of the tubules and the glomerulus (Murphy 2001).
Filoviruses have been ‘officially’ known to science since 1967 however relatively few samples of infected tissue remain available for examination
The pathology of
The kidney contains tubular necrosis and multi focal fibrin thrombi in the glomerular capillaries, resembling disseminated-intravascular-coagulopathy, (DIC) (Figure 26) pulmonary oedema occurs, along side an “effusion of macrophages into the alveoli’ (Murphy 2001). (Figure 27)
In the majority of cases, there is evidence of diathesis in the skin, mucous membranes, soft tissue, visceral organs, stomach and intestines. There may also be swelling of the lymph nodes, kidney, spleen and especially the brain (encephalitis). Focal necrosis is found in the majority of organs, especially in the liver, lymphatic system, testes and ovaries. The liver necrosis is identical to that in Ebola infection. Inclusion bodies are numerous.
Histologic evidence of hemorrhagic diathesis can be found in many organs including panencephalitis and glial nodule formation, perivascular cuffing and interstitial oedema (Murphy 2001).
As with Ebola, there are relatively few samples of
The table below lists the available tests for detection, stating their ease of use, practicality, accuracy and efficiency. (Where applicable)
Advantages – disadvantages.
Indirect immunofluorescence assay (IFA)
Simple to perform but prone to false positives & differential interpretation.
Enzyme linked immunosorbent assay (ELISA)
Specific and sensitive, but initial response is slower than IFA
Immuno - blot
Protein specific – difficult interpretation
Blood, serum & tissue
Rapid & sensitive but requires specialist equipment.
Tissues – skin & liver.
Tissues - liver
Rapid & easy to perform but open to interpretation
Viral nucleic acid - RNA
Blood, serum & tissue
Rapid & sensitive but requires specialist equipment
Blood & tissues
Visualisation of morphology is possible but insensitive & expensive
Blood & tissues
Requires confirmed virus – time consuming
Laboratory diagnosis takes 2 forms, by measurement of the host-specific immunological response to the infection and by detection of viral antigen and genomic RNA in the infected host (Feldmann 1997). The most commonly used assays to detect antibodies are the indirect immunofluorescence assay (IFA), immunoblot and enzyme-linked immunosorbent assays (ELISA) (direct IgG and IgM ELISA, and IgM capture assay). Direct detection of viral particles, viral antigen and genomic RNA can be achieved by electron microscopy (negative contrast, thin-section), immunohistochemistry, immunofluorescence on impression smears of tissues, antigen detection ELISA, and reverse transcriptase-polymerase chain reaction (Feldmann 1997).
No effective treatment exists at present to combat Filovirus infection – no antivirals, no vaccines whether pre or pro phylactic exist, treatment is purely supportive in the form of sedatives and fluid level maintenance. Interferon may be effective in reducing the pace of viral progression, although it is not yet known which form may still be beneficial, as the VP35 protein within the virus of both serotypes contains an anti Interferon function against the alpha 2b form (Leicester 2000).
Interferon was first used therapeutically in the initial 1967 outbreak in Marburg (Leicester 2000), it is unclear whether those which survived did so as a result of Interferon, it would seem appropriate to test any future survivors for increased levels of Interferon to determine whether this is a contributing factor to survival. There are unconfirmed reports of steroids being used to reduce the worse effects of infection. Many possible or theoretical treatments are being explored at present, which will be discussed in the appropriate section.
Filovirus infection primarily targets the Monocytes, (Macrophages) leading to pantropism to all major organs, with a particular affinity for the liver, as this is the location of the recycling process of damaged macrophages. Para-endothelial permeability is the main route of viral progression (Leicester 2000), allowing the virus full, unhindered access to all regions of the body. If this action could be effectively countered, survival rates would be significantly improved however, the present knowledge of the organism does not permit this option.
The present treatment is currently limited to treating the inevitable multiple organ failure and maintaining vital statistics within as close to normal ranges as possible. The first and normally most severely effected organ is the liver and so appropriate liver function tests should be carried out to monitor efficiency. The main enzymes to be observed should be – AST, ALT & AP, along side the accumulation of waste products as an indicator of the severity of the infection.
· Blood sedimentation rates should be closely observed as an increase may indicate necrosis.
· Electrolytes – Sodium, Potassium & Chloride among others. Essential in the osmolarity of various cells and nerve function, electrolyte levels give a perspective on kidney damage.
· Haemodilution – Response to acute blood loss, indicated by the release of immature erythrocytes and high plasma levels
· WBC count – As previously discussed, the virus targets white blood cells, in particular macrophages. WBC counts will therefore be enormously reduced, leading to compromised immunity and secondary infections which should be dealt with accordingly.
Below is a general indication of normal ranges –
4.2 – 5.8 x 1012/L
3.6 -5.6 x 1012/L
13.5 – 17.5g/100ml
11.7 – 15.7g/100ml
39 – 55%
36 – 48%
80.5 – 99.7fL
80.8 – 100fL
26.6 – 33.8pg
26.4 – 34pg
31.5 - 36.3g/100ml
31.4 – 35.8g/100ml
1.5 – 3.5 x 1011/L
1.5 – 3.5 x 1011/L
4.5 – 11 x 109/L
4.5 – 11 x 109/L
1 – 4%
1 – 4%
2 – 8%
Electrical activity in the various organs should also be monitored for any fluctuations - Electrocardiogram, electroencephalogram, electromyogram.
Anti coagulants such as Heparin for example should only be used if there are clear signs of disseminated intravascular coagulopathy (DIC) as this may exacerbate symptoms.
Ribavirin has been tested extensively, but failed to increase survival rates in monkeys (Feldmann 1997) and so it is unlikely to be effective.
Human reconvalesence plasma has been used in conjunction with Interferon, the success rate is unknown.
Individuals who recover from Filovirus infection often suffer prolonged sequelae in the form of orchitis, transverse myelitis, acute hepatitis, uveitis and tunica vasculosa of the eyeball (WHO 2003).
Ease of transmission, high mortality rates and lack of effective vaccines make Filoviruses a level 4 bio-containment organism (Highest level). In the U.K transmission of the virus is most likely to occur in a health care environment – nosocomial, either from the contact of blood, tissue or secretions, or by breathing air borne particles from a cough for example. Individuals at the highest risk are those in close contact with the patient during the period of incubation and illness, ie, direct medical and nursing staff and lab workers handling blood, tissue or other specimens from the patient.
All healthcare systems should be familiar with the guidelines concerning Filovirus protocols, in order to provide appropriate care to patients and to safeguard healthcare workers and the general public, if or when an outbreak occurs.
Standard level 4 procedures should be implemented –
· Installation of Grey zones – Zones between infected areas and the ‘normal’ world, Grey zones should include an area for the adding and removal of protective clothing. A shower dispensing Envirochem or equivalent should be used on entry/exit of infected zone (
· Protective clothing – A Chemturion suit, Racal suit or equivalent must be worn at all times when in infected zone. A minimum of 3 layers of protective gloves must be worn – primary – basic latex gloves, secondary – second layer of latex gloves, plus a third layer of heavy duty biohazard gauntlets. Cuffs should be taped to avoid possible entry points.
· Infected zone should be decontaminated at the earliest convenience, using Envirochem, chlorine or equivalent (National Disease Surveillance Center 2004).
· Sharps – The use of sharps in the form of needles and scalpels etc should be kept to an absolute minimum.
· Only 1 door should remain in use in infected zone, the remaining doors and windows should be sealed and a negative airflow system installed.
· VHF Regional and National response coordinators (
· All suspected cases should be notified to the Director of Public Health and the Director of National disease surveillance (
Hypochlorite solution should be used liberally on to body secretions and fluid for a minimum of 5 minutes prior to disposal. All clothing, soiled linens and general waste should be incinerated in a licensed facility. All sharps should be placed in a rigid yellow sharps bin. Before removal from the infected zone, all sharps bins and other waste containers should be wiped with Hypochlorite solution (NDSC 2004).
Where a case of MHF or EHF is suspected but not confirmed, Ribavirin should be administered whilst awaiting confirmation of diagnosis (Usually through IFA).
The dose and route of administration is as follows –
1. Ribavirin 30mg/kg loading doses, intravenously.
2. 16mg.kg intravenously every 6 hours for 4 days
3. 8mg/kg intravenously every 8hours for 6 days
In cases where a patient has been presented at a general practitioner, arrangements should be made immediately to have the patient hospitalized. The general practitioner has a duty to inform the appropriate people in preparation of the patient’s arrival –
· The consultant microbiologist and laboratory staff
· The infection control team of the receiving institution
· Local public health department
· Emergency incident committee
There should be ongoing close communication between the attending physicians, microbiologists, laboratory workers, the virus reference laboratory, VHF Regional Response co-ordinator and The National Response co-ordinator.
The ambulance should have a police escort to ensure a speedy arrival and to avoid traffic complications.
Filoviruses – Consist of only 2 serotypes, Ebola and
Traditional methods for vaccine development ie – live attenuated virus particles, recombinant proteins and naked plasmid DNA has not been successful against Filoviruses and so alternatives must be explored.
One possible alternative may lie in the recent discovery of the protein which the virus uses to attach to the human cell. The protein destabilizes the cell membrane, allowing the virus entry. It was previously hypothesized that the virus gained entry via phagocytosis. The enzyme Cathepsin B has been experimentally inactivated in human kidney cells, the cells were then infected with Ebola, the virus failed to enter and infect the cells (BBC Online 2005). This is one possible route to developing an anti – Ebola drug however any future drug developed may be accompanied with serious side effects as Cathepsin B is essential in initiating an immune response. The method has not yet been tested on
An extract from the Garcinia kola plant, which is commonly eaten in
1 – Adenovirus containing all 7 viral proteins plus CpG DNA, which has been shown to be important in mediating a strong T cell response and is a strong stimulus to dendritic cells, inducing them to produce high levels of Tl-1 cytokines Il – 12 & Il – 18, which stimulate T cell production. CpG DNA is an effective adjuvant which produces high CTL levels (Brown 2004).
2 – Recombinant plasmid containing viral protein sequences, which could potentially stimulate a fast effective response (Brown 2004).
3 – As above including all known viral strains contained within an Adenovirus vector, this approach has the potential to render an individual immune to all strains.
As previously discussed, Filoviruses only contain 1 surface protein – the glycoprotein. This has been expressed singularly and is effectively an “empty virion’. The glycoprotein produces a moderate humoral immune response and high CTL count (cytotoxic T lymphocyte) in experimentally infected mice. Glycoproteins have also been shown to induce T cell proliferation. 2 months after initial immunization, the mice were found to contain high antibody titres and almost total protection when challenged with Ebola. It has now been concluded that high CTL counts are a contributing factor to survival. This method has also been conducted using the NP protein, the same response was observed only with multiple doses (Feldmann 1997)
Interestingly, when injected with both GP & NP protein bases vaccines, after 1 month of the last injection, antibody titres were significantly reduced (Feldmann 1997)
The glycoprotein based vaccine appears to be a promising possibility, but is still in the trial stages and may be some time before it is widely available.
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 EHF & MHF bears a striking resemblance to Crimean –