It is more than clear to humanity the Darwinian struggle in which the human species has been, and still is, engaged.

Bacterial and viral predators, among others, abound in no more or less abundance than before, and still present a constant threat to individual survival as well as to the prosperity of the human population at large.

With the introduction of antibiotics it was thought that everything would stabilise, but a turning point in medical history would soon be reached, spurred in large part by the global ravages of the human immunodeficiency virus (HIV), which still evades cure or even understanding from the scientific community.

Against these threats is a perpetual backdrop of a multiplicity of infections, which cycle throughout their ecological niches, and are encountered in our modern world to spread with a frightening vigour.

We are currently experiencing the epidemic potential of HIV, but the next epidemic of human disease may be entirely different and almost impossible to predict.

Quoting Donald Henderson of the US Office of Science and Technology Policy: "It is evident now that mutation and change are facts of nature, that the world is increasingly interdependent, and that human health and survival will be challenged ad infinitum by new mutant microbes, with unpredictable pathophysiological manifestations. How are we to detect these at an early stage so as to be able to devise appropriate preventive and therapeutic modalities? What do we look for? What types of surveillance and reporting systems can one devise?"[21]

At the top of the list of urgent threats to public health are new and emerging infectious diseases.

The health of the developed world’s people is inextricably connected to the health of people in other nations around the world.

Infectious diseases disseminate rapidly around the globe, disregarding what one would call geographic barriers, among other barriers. Global surveillance for emerging diseases is vital to public health.

Joshua Lederburg has commented that viruses are humanity’s only real competitors for dominion of the planet, serving as both parasites and genetic elements in their hosts. Not only do they have considerable plasticity, enabling them to evolve in new directions, but their genetic and metabolic entanglement with cells uniquely positions them to mediate subtle, cumulative evolutionary changes in their hosts as well. But they can also decimate entire populations!

The fact that long term natural selection favours mutualism offers only limited encouragement to our species, with millions of people suffering until an equilibrium can be reached.

VHF are one of the greatest concerns due to its propensity to cause incredibly high mortality rates.

The concerns are further increased because even today there isn’t a successful treatment. As so, diseases caused by such viruses are designed quarantinable diseases under the Commonwealth Quarantine Act 108.[29]

VHF viruses come from groups like Arenaviridae, Bunyaviridae, Flaviviridae and Filoviridae. All these groups of viruses contain single stranded RNA genomes.

In the Arenaviridae family, representative members causing haemorrhagic fever are the Junin virus, first identified in 1958 and mainly distributed in Argentina;

Machupo virus identified in 1965, causing Bolivian haemorrhagic fever; Lassa Fever Virus first identified in 1969 in West Africa, and Guanarito virus causing Venezuelan haemorrhagic fever, identified in 1989.

Lassa Fever virus is a single stranded bisegmented RNA virus. It first arose in Nigeria, through a nurse, giving rise to nosocomial transmission.

Nowadays, LFV is a major endemic public health problem, found in all countries of West Africa, and its infection constitutes a significant cause of both morbidity and mortality.

For all theses viruses rodents appear to be the host/reservoir and they are transmitted via excreta. There is another member of this family, Sabiá virus, which caused a haemorrhagic fever in Brazil (São Paulo) and for which reservoir and mode of transmission are yet unknown. Nevertheless, a possible reservoir might be the sabiá, which is the native word for a small simian species present in the Brazilian rainforest, from which the virus has probably acquires its name.

In the Bunyaviridae there are three representative members. One is the Rift Valley Fever Virus, isolated in the 1930s and geographically distributed in Africa, having mammals and mosquitoes as reservoir/vector and body fluids and insect bites as modes of transmission.

Another member is the Crimean-Congo Haemorrhagic Fever (CCHF) virus which is distributed in the South-eastern region of Europe (mainly the Crimea region), Asia and Africa, and is transmitted to humans by the bite of the Hyalomma tick, acting as reservoir and vector, along with some other domestic and wild animals (like ostriches recently!).

The last member is the Hantaan virus that causes the formerly known Korean Haemorrhagic fever. It was firstly observed in 1976 and is now vastly distributed in the Northern Hemisphere. It has rodents as reservoirs and excreta as mode of transmission.

All the viruses mentioned thus far have the potential for human-to-human transmission. However, there are other viruses causing haemorrhagic fever that do not possess this potential, namely Yellow Fever Virus and Dengue Fever Virus.

Dengue Fever virus is a member of the other family causing haemorrhagic fevers, the Flaviviridae. It is an Arbovirus (arthropod-borne virus) and presently the most important insect-borne viral disease in humans.

It is closely related to the occurrence of Yellow Fever, as both viruses share the mode of transmission by using the same mosquito, Aedes aegypti, as a vector. With Dengue Fever other species like Aedes albopictus, Aedes polynesiensis and Aedes scutelbaris have also the potential to act as vectors.

The virus multiplies in the midgut epithelium, brain, fat body and salivary glands of the mosquitoes, causing a "friendly" infection, as no pathological effects have yet been noticed, but it, nevertheless, causes the mosquitoes to be infectious for life.

Dengue Virus replicates in the genital tract of the female mosquito and may enter the ovum at the time of fertilisation, consequently infecting a portion of her offspring.

Treatment studies of Dengue Haemorrhagic Fever have shown that the antiviral Ribavirin is effective in combating the virus in vitro, but for the same action to be achieved in vivo toxic doses of the antiviral would be required.

An effective way of fighting the disease is by controlling its vector.

Mosquitoes can only carry the virus a few hundred feet from where they are hatched, therefore the best way to control the spread of the disease is the elimination of the hatching grounds by emptying sources of stagnant waters (eg. old tires, tin cans...); the use of insecticides and larvicides are also useful as well as the use of mosquito repellents and nets impregnated with insecticide.

Looking at the epidemiology of the virus, there are over three million cases of DHF worldwide, but they are predominantly confined to the Caribbean, Central America and Asia.

Epidemics occur mainly during the rainy seasons, due to increased mosquito breeding sites and hence an increase in mosquito population.

The frequency of the epidemics has increased in the last 15 years, primarily due to increased travel by airplane which provides the mechanism of importation of the virus where Aedes aegypti is not controlled.

The emergence may be due to the expansion of urban populations resulting in increased contact with the vector and increased population densities; the poor water storage facilities and sewage and waste water acting as vector breeding grounds; air travel; ecological changes resulting from urbanisation of rural and other areas; and/or due to an increased population of the mosquito vector, most likely by the development of insecticide resistance.

This disease was first recognised in the 1950s and is today a leading cause of childhood death in some countries.

VHF have clinical similarities and differences.[29]

The non-specific onset of symptoms constitutes a major drawback on differentiating VHF from each other, and furthermore differentiating VHF from other endemic fevers such as Malaria.

Common to some VHF is the timing of the haemorrhagic stage of the disease, which appears during the second week of illness.

VHF are of major concern because of lack of effective specific treatment.

From all VHF, only for LFV there is evidence of effective treatment with Ribavirin.[29] For all the others, and especially Filoviruses, there is no evidence of any available modalities being effective on controlling infection, ultimately causing patients to die.

Filoviridae is a recent taxonomic group of viruses.

Originally classified as Rhabdoviridae, they appear to be more closely related to Paramyxoviridae based on genome sequence data. Nevertheless Filoviridae are distinct enough from all other virus families to guarantee a taxonomic status as a virus family.

It englobes two main virus species: Marburg and Ebola.

The first encounter with any of those viruses was with Marburg virus, in Marburg, a German town, as well as in Yugoslavia in 1967. This first outbreak occurred among workers of a vaccine manufacturing plant, possibly due to handling of African Green monkey tissue imported from Uganda.[8,12]

A similar disease to Marburg appeared in Sudan and Zaire in 1976, but the aetiologic agent proved not to be Marburg virus but a similar virus. This new virus was named Ebola after a river in Zaire, where the outbreak occurred.

Many other outbreaks of Marburg and Ebola virus occurred ever since, with particular incidence of Ebola virus outbreaks, having one of the major and most recent outbreaks had its epicentre in Kikwit, Zaire in 1995.

The virus strains from Sudan and Zaire proved to be different subtypes of Ebola virus.[3,18]

More recently, two other subtypes of Ebola virus have been discovered, Ebola Reston[11] and Ebola Tai.[17]

The first one originated an epizootic in Reston, Virginia in 1989, among a population of monkeys in a research facilities [18]. This strain demonstrated quite different potentials as it apparently did not infect humans, but it also demonstrated the possibility of this strain to be airborne as it seemed to be transmitted through the air in aerosolised secretions. This was suggested because monkeys in separate living quarters contracted the virus without ever coming to direct contact.

The most recent strain discovered, Ebola Tai, discovered on the 24th November 1995, owes its name to the Ivory Coast’s Tai forest, where the virus is thought to have been contracted. This is an interesting strain, as it’s the first time infection of a human has been connected to naturally infected monkeys in Africa.[17]

A Swiss zoologist carried out an autopsy in a dead chimpanzee found in the Tai forest, and she developed typical symptoms of Dengue Syndrome (another VHF). She was hospitalised and recovered without sequelae.

Analysis of blood samples showed negative to presence of antibodies against haemorrhagic fevers such as Dengue, Yellow Fever and the other three strains of Ebola virus.

Later, the pathogen causing the disease has been classified as a member of the Filoviridae, and the latest addition to the Ebola type.

A lot of ecological studies are being carried out in some parts of West Africa in the attempt to find Ebola’s reservoir and/or natural hosts, which until today remains an incognit. The trigger for its re-emergence in new outbreaks in humans has not been identified either.

Dr David Simpson, from Queen’s University of Belfast, has suggested that the fact that infection is lethal to chimpanzees might suggest that they are not the originating source and may have picked up the infection from some other source, possibly some smaller monkey species that they sometimes feed on.[17]

If a virus has only one possible host species then it will probably enjoy limited success by using a strategy of killing its host in a short period of time, hence losing the chance for transmission to new hosts.

Every infectious agent should have some sort of "personal" balance between aggressively replicating and spreading through a host’s tissues, allowing survival of infected hosts long enough for transmission to occur.

The dramatic pathology of Ebola in humans might be due to its poor adaptation to humans as a host.

If Ebola has evolved in a stable relationship with some other host, other than humans and thus humans being accidental in its history, then the limited success of Ebola virus in the human population does not constitute a problem for the virus. The virus will have its "survival" guaranteed by its natural host.

The question still remains and puzzles many scientists in research for an answer. Is there an animal vector, carrier of the virus, possessing immunosuppression to the virus that kills most other primates? Until today no evidence has been put forward, and most arguments and attempts to answer are fruit of speculation.

There is a quite interesting theory that Ebola might have been the cause of a plague in Ancient Athens during the Peloponnesian War.[4]

A Spartan Army had marched through Attica and surrounded Athens in about 431BC. The great Athenian leader Percales ordered the entire population into the city walls. These crowded and often unsanitary conditions became a breeding ground for disease.

During this time one third of the entire Athenian population, including their leader Percales, died.

Thucyclides, the protégé of the historian Herodutus, wrote about this plague, describing some of the symptoms. Whilst many were consistent with Ebola infection, some typical symptoms are notably missed, such as the large quantities of blood usually associated with the infection.

In the "Modern World", Ebola and Marburg have had the following appearances:

• 1967    Marburg, West Germany

                         31 cases, 7 deaths

• 1975    Johannesburg, South Africa

• 1976    Simultaneously in Zaire and Sudan

                        250 cases, 140 deaths

                        Bumba Zone, North Central Zaire

                        318 cases, 290 deaths

• 1977    Tandala, Zaire

• 1980    Frenchman dies of Marburg in Kenya
• 1987    Danish boy dies of Marburg in Kenya
• 1989    Reston, Virginia, USA

• 1989    Sienna, Italy

• 1995    Kikwit, Zaire

                         77% mortality

• 1995    Gozon, Ivory Coast
• 1996    Plibo, Liberia

• 1996    Mayibout, Gabon

• Apr1996Alice, Texas, USA

• 1996    Gabon

• Oct 1996Johannesburg, South Africa

                            Ebola imported from Gabon by a doctor seeking medical attention. Proved fatal to a nurse.

Ebola virus haemorrhagic fever is a severe, systemic, febrile illness caused by infection with Ebola virus.

After a variable incubation period of 2-21 days[32], there is onset of high fever (103-104^oF), headaches, myalgia and sore throat. These are non-specific clinical symptoms and occur in the first phase of the disease.

One to three days after, patients are prostrated with diarrhoea and vomiting, abdominal pain and nausea and usually present are also pharyngitis and conjunctivitis, lymphadenopathy and limited liver and kidney malfunction.

Diarrhoea and vomiting usually have serious consequences on the patient, being sometimes the cause of death by dehydration, i.e. death by intractable shock.

After 5-7 days, a transient, nonpruritic maculopapular exanthematous rash appears.

Patients rapidly develop chest pains, cough, dysphagia, jaundice, pancreatitis and central nervous system disfunctions (somnolence, delirium or even coma).

Also at this time, in a large proportion of infected people, severe spontaneous bleeding starts in many organs, in the skin, nose and gums, and from mucous membranes and needle puncture sites.

During the second week of illness, patients defervesce, improve or die. Death can be due to multiorgan failure, hypovolaemic shock (consequence of haemorrhage and dehydration) or disseminated intravascular coagulation.

Another important event occurring 10-14 days after infection with Ebola virus, i.e. two weeks after infection, is that humoral immune response can be detected. This immune response consists of antibody production directed primarily against surface glycoprotein in Ebola virus. However, this immune response generated is very ineffective.

From all the symptoms Ebola virus infection can cause in humans, there are some, crucial for the correct diagnosis of infection by Ebola virus. These are laboratory findings of thrombocytopenia, leukopenia, neutrophilia and elevated transaminases as well as clinical findings of diarrhoea, abdominal pain and maculopapular rash.

Both Marburg and Ebola viruses are pantropic, i.e. they infect and cause lesions in many organs of the human body, but especially the liver and spleen become enlarged and dark.

Histopathological change caused especially by Ebola virus, includes focal hepatic necrosis, with little inflammatory response, follicular necrosis of lymph nodes and spleen and also focal necrosis in other organs such as kidneys, ovaries and testicles.

In late stages the central lesions appear to be those infecting the vascular endothelium and platelets. The manifestation of this is the occurrence of haemorrhage in the gastrointestinal tract, pleural, pericardial and peritoneal spaces and into renal tubules with deposition of fibrin.

Abnormalities in coagulation parameters include fibrin degradation products (FDP) and prolonged prothrombin and partial thromboplastin times, suggesting that disseminated intravascular coagulation is a terminal event.

However, in the Reston strain, viral replication was extensive in fixed tissue macrophages, interstitial fibroblasts of many organs, circulating macrophages and monocytes, and less frequently in vascular endothelial cells, hepatocytes, adrenal cortical cells and renal tubular endothelium. As such, macrophages and fibroblasts seem to be the initial and preferred site for Ebola Reston replication.

The virus is usually isolated from acute phase sera, but it has also been found present in throat washes, urine, soft tissue effusates, semen and anterior eye fluid, even when the specimens were obtained late in convalescence.

Virus has also been typically isolated from post-mortem material, especially from tissue provenient from organs like spleen, lymph nodes, liver and kidneys.

It is worthy mentioning that the virus has rarely been isolated from brain or other type of nervous tissue.

The mode of primary infection in any natural setting is yet unknown for both Marburg and Ebola viruses. All the secondary cases have been nosocomial or caused by intimate contact with the patient.[29]

The virus spreads mainly through the blood and replicates in many organs.

Transmission occurs through direct contact with blood, organs or semen of infected people.

Interestingly, sexual transmission, through semen, carries on for longer than the others. In some documented cases, virus has shown to be present in the genital secretions of patients who recovered from the illness, for a period of about 7 weeks after clinical recovery.

The major factor in nosocomial transmission is the combination of the unawareness of the possibility of the disease by a "worker", who is also inattentive to the requirements of effective barrier nursing after diagnosis. This causes nosocomial transmission risk to be small. Nevertheless, in the 76 epidemics in Zaire, every Ebola case was caused by contaminated hypodermic syringes, which have been re-used without sterilisation in a missionary hospital.

This normally occurs in countries where the health care system is underfinanced.

Another type of transmission has been put forward at the time of the 89 Reston outbreak. Also experimental aerosols generated in the laboratory have indeed infected monkeys, but there is no evidence for inter-human transmission of Ebola virus by aerosols.[11]

Association with bats has been implied in at least two episodes of Ebola, when individuals entered the same bat-filled cave (Kitum Cave) in Eastern Kenya.

Ebola infections in Sudan in 1976 and 1979 occurred in workers of a cotton factory containing several bats on the roof. But in all cases, study of antibody in bats has failed to detect evidence of infection, and isolation of virus from bat tissue has never been successful.

To sum it up, epidemics normally result from person to person transmission, nosocomial spread or laboratory infections.

As the primary mode of person to person transmission is the parenteral route, i.e. due to contaminated blood, and through secretions or body fluids, it is very important to keep under strict surveillance (body temperature checked twice a day, with immediate hospitalisation and strict isolation in cases of temperatures above 38.3^oC) people who have had close physical contact with patients.

Casual contacts should not be dismissed, they should be put on alert and asked to report any abnormal hyperthermias. Surveillance of suspected cases should continue for at least three weeks after the date of the last contact. Additionally they should be isolated from the other patients and strict barrier nursing techniques practised.

Particular care should be taken to make sure high risk procedures, like placing the intravenous lines and the handling of blood, secretions, catheters and suction devices, are done under barrier nursing conditions.

Furthermore, hospital staff should have individual gowns, masks and gloves, and gloves and masks must not be re-used unless disinfected.

Handling of patients who died from the disease should be prompt. Ideally they should be cremated, but traditional burial is also effective.

There is no specific treatment or vaccine for the infection with these viruses.

Nevertheless experimental treatments have been tested and these include the use of human interferon, human convalescent plasma and anticoagulation therapy.

The use of interferon inducers (eg. ridostin), interferon systems (eg. reaferon) have no significant effect on longevity.

Immunisation of horses, sheep and goats are presently used to develop antibody preparations for treatment of Ebola infected individuals.

These treatments have come up with mixed results and any success that might have arisen is controversial.

The only recommended "treatment" by the World Health Organisation (WHO) is isolation and supportive care of patients.

The viruses have also shown to be resistant to in vitro action of the antiviral drug Ribavirin[29]. Therefore, the only effective preventive measure currently known relies on the creation of a physical barrier, capable of preventing transmission of the virus to uninfected people.

Ebola and Marburg are highly transmissable viruses and thus present a potential danger to society.For this and other reasons they have been considered Biosafety Level 4 pathogens, and hence some important security measures must be taken.

High security isolation is necessary and must be supplemented with high security laboratory services.

Issues that must be addressed include construction, ventilation, filtration and humidity, together with protective

measures for staff and careful handling of laboratory specimens.

Materials or vehicles the patient has had contact with will have to be decontaminated.

Four products are highly effective in decontamination procedures: sodium hypochlorite, gluteraldehyde,

formaldehyde and ethylene oxide.

Containment procedures ideally include two anti-rooms to patient chamber, which are used to decontaminate

health care workers before and after patient contact.

The patient chamber should also have negative pressure.

Ebola and Marburg viruses are quite infective at room temperature, but when heated to 60^oC and kept for 30 minutes, the virus will lose its infectivity and hence be inactivated.

There are other forms of inactivation of these viruses. They can be inactivated by irradiation with ultraviolet (UV) light, or g -radiation (gamma), 1% formalin, b -propiolactone, and brief exposure to phenolic disinfectants and lipid solvents, like deoxycholate and ether.[10,16]

Detection of virus particles are easily visualised by EM techniques, during viraemia, therefore evidencing presence of virus particles in the blood and consequently assuring infection is taking place.

Serum antibody titres can be determined by direct immunofluorescent assay (IFA) carried out on a microscopic slide containing a monolayer of infected cells.

IgG and/or IgM specific immunoglobulin assays are then performed. The specific reactivity of positive sera should be confirmed by either radioimmune precipitation or Western Blot analysis.

The ELISA technique was used by the CDC personnel for rapid, simple and on-site testing using Ebola antigens to confirm the presence of the virus.

Filoviruses are morphologically identical to Rhabdovirus particles, although they are much longer.

The virions are highly pleiomorphic and filamentous, sometimes appearing as branched or spheroidal (U-shaped, 6-shaped or circular) forms.[10,16]

Virions vary greatly in length, and they can be up to 1400nm long (790-970nm long after purification), but have a uniform diameter of 80nm.[10]

Purification by ratezonal gradient centrifugation showed that Marburg virus has its peak infectivity at 665nm length and Ebola virus at 805nm, and both have a bacilliform outline.[16]

Despite the discrepancy in length, filoviruses seem to be quite similar in all other morphological aspects.

They consist of a nucleocapsid which is a dark central axis 20nm in diameter, within which there is an axial channel about 10-15nm.[10]

These are in turn surrounded by a helical nucleocapsid of 50nm, bearing cross-striations with a periodicity of approximately 5nm.[10]

The helical nucleocapsid is surrounded by a lipoprotein unit membrane, from which 7nm spikes consisting of a viral encoded protein with a carbohydrate decoration, protrude through the membrane, and are dispersed at approximately 10nm from each other. [10]

Virus particles have a molecular mass of approximately 4*10⁶, and a density determined by centrifugation through a 10-40% potassium tartarate gradient of 1.14g/ml.[14]

Uniform particles have a sedimentation coefficient of 1300-1400S.

The genome of the filoviruses consists of a molecule of linear, non-segmented, negative polarity, single stranded RNA. [24]

Without its "tubular armour" the viral RNA would be non-infectious and rapidly broken up by ubiquitous RNase enzymes.

The RNA strand is not polyadenylated and is complementary to viral specific messenger RNA.

The genome amounts 1.1% of total virion weight and the sedimentation coefficient is 46S (0.15M NaCl, pH7.4).[10]

The RNA molecule is about 19000 monomer units long and very rich in adenosine and uridine residues.

The genome shows a linear arrangement of genes in the order:

Each gene of the negative stranded RNA is flanked by control sequences directing the independent copying of each gene to a separate message [26]. These are highly conserved start and termination signal sequences located at the 3’ and 5’ ends of the gene respectively.[26]

They all contain the pentamer 3’ UAAUU 5’.

The genes are either separated by intergenic regions variable in length, but usually larger than 94bp, and also variable in nucleotide composition; or by gene overlaps which are limited to the conserved transcription signals centred around the common pentanucleotide sequence.

Ebola viruses show three overlaps that alternate with intergenic sequences, while Marburg virus only contains a single overlap and at a different position.

Extragenic "leader" sequences are present at either ends of filovirus genomes and they are complementary at their very extremities. These sequences are also found in other nonsegmented, negative stranded RNA viruses (Mononegavirales order).

The viral genome codes for 7 different viral proteins. Four proteins associate in the ribonucleoprotein complex (NP, Polymerase, and structural Proteins 30 and 35). There is also a glycoprotein and two other structural proteins.[2,13,14,16,23,28]

Detailed analysis of proteins will be looked at further along.

The mechanism of entry of Filoviruses into the cells is still unknown, but it is reasonable to think that, in analogy to Rhabdoviruses and Paramyxoviruses, the glycoprotein, as the only transmembrane protein of the virion particle, mediates the adsorption and the penetration processes.

Transcription and replication take place in the cytoplasm of infected cells, which will develop prominent inclusion bodies containing viral nucleocapsid proteins.

The Filovirus genomes are transcribed yielding monocistronic subgenomic messenger RNA species which are complementary to viral genomic RNA.[26]

The 5’ ends of the subgenomic RNAs start at the transcription start signal sequence, and the 3’ ends carry a poly (A) tail generated by the polymerase at a run of uridine residues located at the 5’ends of all transcription termination signal sequences.[16]

Replication of the genome is mediated by synthesis of full-length complementary antigenome (positive sense) which serves as a template for the synthesis of progeny negative-strand RNA anticomplementary to the parental RNA.[23]

The complementarity of the genome extremities suggest that a single, identical encapsidation site on the genome and antigenome exists and an identical entry site for the polymerase in both transcription and replication modes.

RNAs have the potential to form stem and loop structures that might may play an important role in gene expression (perhaps by altering ribosome binding). The hairpin shape of the leader strand may be conductive to nucleoprotein binding, and the subsequent conformational change may either promote replication or transcription.[22]

The cytoplasmic inclusion bodies grow and become highly structured as the infection proceeds. They culminate with the release of entire viral particles, by releasing a lytic enzyme that cuts a hole in the membrane, allowing the virus to escape due to an increased sensitivity to osmotic changes.

The virions have seven different viral encoded proteins, which apparently have identical functions for the different viruses.

The molecular weight of each protein has been deduced and they are different for Marburg and Ebola viruses:

The nucleoprotein and VP30, which has been considered a minor nucleoprotein, are tightly associated with the ribonucleoprotein (RNP) complex. These two proteins are the only two phosphorylated from the seven proteins encoded by the virus.[5]

Loosely associated with the RNP complex are also the L or polymerase and VP35.

VP35 seems to be a component of the transcriptase complex, analogous to the P protein of Paramyxoviridae and the NS protein of Rhabdoviruses. This is further supported by experiments showing its release from the RNP complex by increasing salt concentration.

The L protein, the virion encoded RNA-dependent RNA polymerase, is the largest protein encoded by the viral genome.[5]

VP40 and VP24 functions are yet unknown. It is thought that they are membrane components and that due to its weak association with the RNP complex, VP40 is a matrix protein. VP40 has a net positive charge.

VP40 was thought to be a nucleocapsid associated protein, but this is probably because of contamination, as VP40 is the most abundant protein of all virus proteins. Further studies have suggested that it is in fact a component of the virion membrane, in the layer between the RNP core and VP24 protein. This shows that the material enclosing the nucleocapsid is multilayered[5].

The nucleoprotein gene is the first to be transcribed. A Kyte Doolittle analysis of the predictive amino acid sequence yields a hydrophobic N-terminal and a hydrophilic and highly acidic C-terminal regions. The protein has a net negative charge.

The glycoprotein is an integral membrane proteins and forms the surface projections of the virion. This is the protein thought to be responsible for mediating the entry of the virus into the host-cell. Functional sites for receptor recognition and binding should, therefore, be located on the glycoprotein.

The glycoprotein of Marburg virus is a type I transmembrane protein, and inserted in the lipid envelope as a homotrimer.[7,16]

The carbohydrate structures of this protein account for probably more than 50% of the total molecular weight of the mature protein. The types of glycosylation found in this protein include oligomannosidic and hybrid type N-glycans, as well as bi-, tri-, and tetra-antennary complex species, and high amounts of neutral mucin-type-O-glycans.[7]

Lectin binding studies showed N-and O-linked oligosaccharide structures are also found in Ebola virus. The only difference on the carbohydrate structures of the glycoprotein is the Ebola glycoprotein is terminally sialated, whereas Marburg virus glycoprotein totally lacks terminal sialic acid residues. [7]

The glycoprotein gene contains a translational stop sequence in the middle, which results in the premature termination of the glycoprotein[31]. This shorter protein is secreted and has no apparent function, and in fact is not tolerated by the infected cells. To overcome this problem, the virus adopts a strategy by which the glycoprotein gene is edited, allowing the joining of two ORFs and hence bypassing the stop sequence and consequently translating the full-length, fully functional glycoprotein.[31]

The mRNA editing event is analogous to the one occurring in Measles virus. What happens specifically is the addition to the subgenomic mRNA molecule of a non-templated adenosine (A) residue, resulting in frame-shifting.[31]

It has been shown that not only Ebola virus RNA-dependent RNA polymerase recognises the glycoprotein mRNA editing site, but also DNA-dependent RNA polymerases of different origins, such as the vaccinia virus polymerases.[31]

The glycoprotein of the Filoviruses contains a sequence of 26 amino acids in the C-terminal region, which shows an 80.9% homology between Marburg and Ebola viruses.

Furthermore, this particular stretch also shares homology with the same protein of oncogenic retroviruses.

These findings provide some insight on the pathogenesis of the filoviruses, as synthetic peptides of the GP gene yield a highly compromised immune system, leading to the inhibition of the blastogenesis of lymphocytes, a reduced chemotactic ability of monocytes and macrophages, and the inhibition of natural killer (NK) cells.

The glycoprotein includes a variable hydrophilic central region flanked by hydrophobic regions containing most of the cysteines used in disulphide bridges.

Protein synthesis rates appear to be different between the Zaire and the Sudan strain of Ebola virus.

Ebola Zaire proteins can be found after 24 hours, whereas Ebola Sudan requires a longer period of time.

Ebola proteins are first seen at 6 hours post infection, being the NP and GP the first to appear, followed by VP35 at 8 hours post infection.[5]

The time course studies of protein synthesis show that the synthesis of the more abundant protein, VP40, lags behind the synthesis of the less abundant protein, VP35.

This finding suggests that VP35 might play a somewhat important role in the early stages of the infection.[5]

Theoretically, RNA viruses have a higher potential for rapid evolution due to the use of high error rate RNA polymerase enzymes for their replication.

Nevertheless there is a misconception about the stability of RNA genomes in nature. Most RNA viruses remain very stable when confined to their native ecological niche.

Analysis of two Ebola Zaire strains (Zaire ‘76 and Kikwit ‘95) showed that the virus patent in outbreaks has had minimal or even negligible evolution over time. Several comparisons of the viral genome of both strains have been made, and there is little change evidenced.

For instance, the glycoprotein gene of 528 base pairs differs in 4 base pairs (<1%) and no differences in the Polymerase gene of approximately 350 base pairs, within 48 hours. Three days later, the entire genome differed in less than 1.6%.

There is little change patent, over 19 years and in extreme ends of Zaire, in the genome of Ebola virus (Sanchez et al., 1992).

This finding leads us to the thought that Filoviruses in general (for which Ebola has been used as a model) are exquisitely stable and have evolved to occupy special niches in the wild.

An inevitable consequence of the low fidelity polymerases used by RNA viruses, is the generation of a large population of different viral genomes, often designated as quasispecies.

So, among their hosts, RNA viruses tend to be transmitted as quasispecies rather than single clones.

Given a host where the virus is confortable, genetic drift is minimal; viruses rarely encounter a bottleneck that might trigger significant drift. Thus, in the absence of such bottleneck it is highly unlikely that RNA quasispecies will change "en masse". Only nonsynonymous mutations are likely to undergo substantial selection, and in long term the major part of these changes have proved to be deleterious to the virus.

Furthermore, nucleotide changes that do not affect the coding capacity may also have an important effect on other functions of viral RNA.

For all the negative stranded RNA viruses the genome serves more than just a source of information for the synthesis of viral proteins. The genome is a vital part of the virus structure.

Transcription does not use viral genome as a template but an association of the genome and the nucleoprotein, combining in what is designated by ribonucleoprotein (RNP) complex.

The way the RNA interacts with the nucleoprotein is strongly influenced by the nucleotide sequence of the genome. In all, mutations that are apparently silent, based on coding capacity, will be greatly repercuted in the structural organisation of the virus particles.

Constraints in viral evolution are not surprising when we consider the selective pressures imposed by the host at every stage of the virus life cycle.

It seems likely that the stability of Ebola virus is merely a reflection of the selection pressures exerted on the infectiousness of progeny virions.

Genomes are shaped by their host systems, and host selects variants in transmissibility and other properties.

Tissue tropism determinants include the site of entry, viral attachment proteins, host cell receptors, tissue-specific genetic elements (like promoters), host cell enzymes (proteinases), host transcription factors and host resistant factors like age, nutrition and immunity.

Viral virulence is very different from bacterial virulence. Whereas bacterial virulence is largely mediated by bacterial toxins and virulence factors, viral virulence is dependent on host factors.

Because virulence is multigenic, defects in almost any viral gene may attenuate the virus.

Interaction of the virus and its host is complex but highly ordered.

They can co-exist in equilibrium until environmental changes shift the equilibrium forcing rapid evolution of the virus.

It is reasonable to expect that new viruses will occasionally emerge, but the stochastic and multifactorial nature of viral evolution (especially from RNA viruses) difficultates the prediction of when such events will occur.

Compared to other viral haemorrhagic fevers, like Dengue or Yellow Fever, Ebola and Marburg present a bigger threat as no host or vector is known, and hence it becomes much more difficult, or even impossible, to compile an epidemiological history and prediction of where and when an outbreak might occur. With Dengue it is reasonable to expect higher incidence during the rainy seasons due to increased grounds of breeding for the mosquito-vectors, which in turn enables the population to be prepared to fight the carriers of the virus, hence reducing the transmissibility and preventing the spread of the disease.

With Ebola and Marburg we are "kicking" in the dark. There are no climacteric changes that might lead us to an outbreak, nor there is a particular vector that one can fight against, in order to decrease the spread of the disease.

Filoviridae, as RNA viruses, demonstrate an unusual stability in the wild, being able to remain "dormant" in a yet unknown mechanism and/or host, re-emerging unpredictably, showing practically no genetic variance, suggesting that somewhere in the wild, there must be a way by which the viruses remain hidden, conferring them an unusually high degree of stability.

Whilst molecular studies on the viruses have been successful, epidemiological studies strongly failed to show any relevant results. These could reveal to be very important in managing the disease and possibly think of eradication.

Meanwhile, these viruses present an important threat to mankind, especially in less developed countries where outbreaks have been constantly occurring. Scientists are unable to predict where and when the next epidemic might be, as a lot, or probably most of the Ecology of the virus still remains an Enigma to everybody.

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