Wednesday, September 13, 2006

Filoviruses




The Filoviruses (Latin – Filo – Threadlike) are a relatively new family of viruses, consisting of only 2 known serotypes – Ebola virus and Marburg virus. Transmission to humans is rare and as yet not fully understood, although it has been hypothesized that the virus may be transmitted through air borne particles and compromised epitheliales.

The Filoviridae family has been known to science since 1967 when an outbreak of hemorrhagic fever infected 31 lab workers in Marburg, Frankfurt, resulting in 7 deaths (DM Sander 1993). These workers had been exposed to tissue from African green monkeys (Cereopithicus aethiops) imported from Uganda. Subsequent outbreaks have occurred sporadically

. The largest Marburg haemorrhagic fever outbreak (excluding the current outbreak in Uige Angola which is discussed later) was recorded between 1998 and 2000 in North-eastern Democratic Republic of Congo where there were 150 confirmed cases and 63 fatalities (Center for Disease Control 2001).

The infrequency of Marburg outbreaks is not a reflection of our control of the virus but rather a measure of its affinity to a human host.

In 1976 a new threat emerged in Zaire and Sudan simultaneously, there were 500 confirmed cases which resulted in 460 deaths (World Health Organisation 2003), a mortality rate of 92%.

This was the emergence of Ebola Hemorrhagic fever. So called after the river in the Democratic republic of Congo where it was first discovered. (Or more accurately in a cave in Mount Elgon – Uganda) It would appear that the Ebola virus is generally the more virulent of the 2 serotypes. Kikwit, Zaire is home to the most recent large scale epidemic (1995) which claimed 233 lives from 293 confirmed cases (Mediscover 2002). This virus is genetically very similar to the initial case in 1976. Like the Marburg virus, Ebola virus tends to appear, infect all available hosts and disappear as mysteriously as it arrived, or so it would seem. Its natural reservoirs are as yet unknown, making an accurate calculation of its true origins virtually impossible.

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.

Filoviruses are unusual in that their genome has remained relatively unchanged since their discovery – Marburg has no known subtypes & Ebola has 4. The virus responsible for the recent outbreak in Kikwit, Zaire differs only by 1.6% to that of the Ebola Zaire virus of 1976 (CDC 2001). This would suggest that Filoviruses are very host specific, ie only able to infect a limited number of species. Filoviruses thrive in warm humid conditions, especially those of central Africa, further limiting their range. When conditions are ideal, the ‘Red Death’ strikes with such sudden ferocity and venom that local authorities are powerless to react accordingly, failing to provide adequate isolation and care, resulting in many fatalities

Distribution –

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

Areas of Marburg virus distribution are found in the drier areas – Eastern and South central Africa, however the accuracy of this survey may be lacking due to the infrequency of Marburg occurrences. The pattern of distribution of the 2 Filovirus serotypes would suggest that the host species have quite different ecologic requirements.



Ebola Reston - Ebola Reston was first discovered in Reston, Virginia, where monkeys (Cereopithicus aethiops) imported from the Philippines where found to be infected. There are no records of Ebola Reston virus in the Philippines, although environmental conditions which support Filoviruses in Africa occur in the Philippines and other Asian regions.The geographic origins of E.Reston remains unknown. Despite ample opportunity, E.Reston does not appear to be capable of human infection (CDC 2001).

Epidemiology & Transmission –

Marburg & Ebola are (or would appear to be) indigenous to the African continent. Ebola has only been isolated from individuals in or from Africa (Feldmann 1997).

Marburg has been isolated from individuals in both Africa and Europe, although all have their origins in Africa.

Ebola Reston has been detected in captive non-human primates in Asia by means of IFA (Indirect fluorescence antibody) testing.

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 Germany, USA and the Philippines (Feldmann 1997).

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 Zaire (International Committee for the Taxonomy of Viruses 2002).

Transmission from droplets and airborne particles has been observed experimentally in infected monkeys.

In September 1983, Frederick, USA, Dr Eugene Johnson of the United States Army Medical Research Institute of Infectious disease carried out experiments looking into possible treatments for Filovirus disease. In 1 particular experiment he infected 12 African green monkeys with the most virulent strain of Ebola virus – the Mayinga strain of Ebola Zaire (92% mortality), 2 were used as controls, ie no Ebola injection. Various treatments were used to aid recovery, none were successful. All injected monkeys died, the 2 remaining ‘controls’ stayed healthy, but later tested positive and subsequently died, apparently from airborne transmission. All monkeys tested negative before the commencement of the experiment. (Preston 1995) This was the discovery that Ebola Zaire could be airborne

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 (Preston 1995).

Fatal Filovirus occurrences –

Virus

Country

Origin

Date





Ebola Ivory Coast

Cote d’ Ivoire

Tai national park

Nov 1994

Ebola Ivory Coast

Cote d’ Ivoire

Plibo (Liberia)

Dec 1995

Ebola Sudan

Uganda

Gulu

Oct 2000-01

Ebola Sudan

Sudan

Nzara

Nov 1976

Ebola Sudan

Sudan

Nzara

Oct 1979

Ebola Zaire

DRC

Yambuku

Sept-Oct 1976

Ebola Zaire

DRC

Bonduni

June 1977

Ebola Zaire

Gabon

Minkebe

Dec 1974

Ebola Zaire

DRC

Kikwit

Jan-Jul 1995

Ebola Zaire

Gabon

Mayibout

Feb-1996

Ebola Zaire

Gabon

Booue

Jul-1996-Mar-1997

Ebola Zaire

Gabon & DRC

Ekata

Dec-2001-02

Marburg

Zimbabwe

?

Feb-1975

Marburg

Kenya

Mt.Elgon

Jan-1980

Marburg

Angola

Uige

Oct-2004-Present

Ebola Zaire

DRC

?

Feb-2003

Marburg

Kenya

Mt.Elgon

Feb-1987

(CDC 2001)

Recent outbreaks –

October 2004 saw the start of an outbreak of Marburg in Uige Angola. The death toll presently stands (as of 05/05/05) at 271 from 313 confirmed cases (Pharmiweb 2003), an unusually high mortality rate of 86%. Such a high death rate would normally be associated with Ebola Zaire which often kills around 92%. Marburg usually kills 1 in 4 (CDC 2001). The outbreak is not yet under control and deaths are occurring daily. This is the largest single occurrence of Marburg virus (CDC 2001). There is no history of Marburg in Angola.

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 Zaire.

Pathogenesis-

Infection and the activation of Monocytes -

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.

Symptoms –

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).

ClassificationandStructure –

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. Marburg is as yet unique and has no known sub-types. Ebola has 4 sub-types – Sudan, Ivory Coast, Reston and Zaire. Genomic analysis of the genomes demonstrates a close relationship to Rhabdoviridae & Paramyxoviridae. All 3 families make up the order Mononegravirales (CDC 2001).

Comparisons of Ebola and Marburg

Marburg (Lake Victoria)

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.

Marburg appears shorter and broader, often with a ‘shepherds crook’ at one end.

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. Marburg – 19,100) (DM Sander 1993)

NP –Nucleoprotein.

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’

(PubMed 2002)

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.

Replication –

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.

The Marburg GP is expressed in a single frame, the gene does not contain sequences with mechanisms such as editing or frame shifting (Feldmann 1997).

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)

Overview -

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 (Preston 1995).

Ebola infection -

Liver samples from Ebola and Marburg infection are strikingly similar, ie all show fatty change and necrosis of the hepatocytes and Kupffer cells (Murphy 2001). The necrosis is normally distributed throughout the lobules, from the central veins to the lobular peripheries. Darkening of the nuclei (nucleoclasia) often occurs, (As can be seen in the header photo) rarefaction, cytolysis and swelling occurs, spilling cell contents into the surrounding tissue, encouraging the cascade effect (WHO 2003), however little inflammatory infiltration into the sinusoids occurs, unusual considering the extent of the necrosis (Murphy 2001). High numbers of hepatocyte mitoses are observed, but these are rapidly overcome by the same necrotic changes as they become encapsulated by the expanding foci of infection.

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

Marburg infection –

The pathology of Marburg and Ebola infections show exact similarities, hepatocellular necrosis (figure 20) is the most prominent effect.(Murphy 2001). At the point of death, varying states of cytoplasmic hyalinization (Conversion into Hyalin – product of amyloid degeneration) and eosinophilia can be observed. Inclusions and Councilman like bodies are often found in the liver tissue.

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 Marburg infected tissue –

Laboratory diagnosis –

The table below lists the available tests for detection, stating their ease of use, practicality, accuracy and efficiency. (Where applicable)

Test

Target

Source

Advantages – disadvantages.

Indirect immunofluorescence assay (IFA)

Antiviral antibodies

Serum

Simple to perform but prone to false positives & differential interpretation.

Enzyme linked immunosorbent assay (ELISA)

Antiviral antibodies

Serum

Specific and sensitive, but initial response is slower than IFA

Immuno - blot

Antiviral antibodies

Serum

Protein specific – difficult interpretation

Antigen ELISA

Viral antigen

Blood, serum & tissue

Rapid & sensitive but requires specialist equipment.

Immunohistochemistry

Viral antigen

Tissues – skin & liver.

Time consuming

Fluorescence assay

Viral antigen

Tissues - liver

Rapid & easy to perform but open to interpretation

PCR

Viral nucleic acid - RNA

Blood, serum & tissue

Rapid & sensitive but requires specialist equipment

Electron microscopy

Viral particle

Blood & tissues

Visualisation of morphology is possible but insensitive & expensive

Virus isolation

Viral particle

Blood & tissues

Requires confirmed virus – time consuming

(Feldmann 1997)

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).

Treatment –

Present treatment –

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.

Maintenance of vital ranges –

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.

Observations –

· 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 –

Parameter

Men

Women

RBC Count

4.2 – 5.8 x 1012/L

3.6 -5.6 x 1012/L

Haemoglobin

13.5 – 17.5g/100ml

11.7 – 15.7g/100ml

Haematocrit

39 – 55%

36 – 48%

MCV

80.5 – 99.7fL

80.8 – 100fL

MCH

26.6 – 33.8pg

26.4 – 34pg

MCHC

31.5 - 36.3g/100ml

31.4 – 35.8g/100ml

Platelets

1.5 – 3.5 x 1011/L

1.5 – 3.5 x 1011/L

WBC

4.5 – 11 x 109/L

4.5 – 11 x 109/L

Neutrophils

60%

60%

Eosinophils

1 – 4%

1 – 4%

Lymphocytes

30%

30%

Monocytes

2 -8%

2 – 8%

Basophils

<1%

<1%

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).

Safety precautions for hospital & laboratory staff.-

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.

Protective clothing and effective maintenance of infected zones –

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 (Preston 1995).

· 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 (UK) should be kept informed of any developments which may result in exposure to the general public (NDSC 2004).

· All suspected cases should be notified to the Director of Public Health and the Director of National disease surveillance (UK).

Clinical waste management –

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).

Preliminary diagnosis and patient transport –

Where a case of MHF or EHF is suspected but not confirmed, Ribavirin[1] 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

(NDSC 2004)

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

(NDSC 2004)

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.

Current research –

Filoviruses – Consist of only 2 serotypes, Ebola and Marburg. Both Ebola and Marburg pose a serious threat to humans and are extinction level viruses. Ebola and Marburg are arguably far more dangerous than HIV which has claimed the lives of millions since its discovery in the late seventies. HIV destroys the immune system absolutely in approximately 10 years. Filoviruses can do this in 10 days. Both have their origins in Central Africa

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 Marburg.

An extract from the Garcinia kola plant, which is commonly eaten in Western Africa, has been extremely effective in halting viral replication in laboratory testing and is currently undergoing further trials (BBC Online 2005).

Accelerated vaccines –

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.

‘Empty’ virion approach –

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.

References

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http://news.bbcco.uk/1/hi/health/medicalnotes/ - Accessed – 11/05/05)

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http://www.tulane.edu/-dmsander/www/335/filoviruses.html - Accessed 11/05/05

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http://www.gsbs.utmb.edu/microbook/ch0724408289.stm.htm - Accessed 11/05/05

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http://www.itg.be/ebola/ebola-17.htm - Accessed 16/05/05

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http://www.cdc.gov/ncidod/dvrd/spd/mnpages/dispages/filoviruses.htm - Accessed 16/05/05

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http://www.asm.org/searchresults.asp - Accessed 18/05/05

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http://www.ndsc.ie/publications - Accessed 18/05/05

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http://www.ncbi.nlm.nih.gov/genomes/framik.cgi?db=genome&gi=10467 – Accessed 20/05/05

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http://www.avytal.co.uk/features/feature.asp?row_id=578 – Accessed 21/05/05

JVG 2002 – Journal of General Virology (Online) Available at –

http://www.sgm.ac.uk/jgvdirect/17772/17772ft.htm - Accessed 21/05/05

Peterson 1997 – Center for Disease Control (Online) Available at –

http://www.cdc.gov/ncidod/eid/vol10no1/03-0125.htm Accessed 21/05/05

Mediscover 2002 – Mediscover.com (Online) Available at –

http://www.mediscover.net/clipsdet.cfm?clippingsid=257 – Accessed 21/05/05

WHO 2003 – World Health Organisation (Online) Available at –

http://www.who.int/ebola/ebola - Accessed 21?05/05

Bioscience 1999 – Comparing Ebola and Marburg (Online) Available at -

http://www.bioscience.drexel.edu/homepage/oldcourses/ebe5/webprojects/ - Accessed 21/05/05

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Brown 2004 – DNA based vaccine (Online) Available at –

http://www.brown.edu/courses/bio_160/projects2004/ebola/infection.html - Accessed 22/05/05



[1] EHF & MHF bears a striking resemblance to Crimean – Congo VHF & Lassa fever symptoms in the early stages and so Ribavirin should be administered as a precautionary measure whilst awaiting results. Filoviruses do not appear to be susceptible to Ribavirin.