Taxonomic Structure of the Family
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Order |
Mononegavirales |
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Family |
Filoviridae |
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Genus |
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Genus |
Virions are pleomorphic but have mainly bacilliform or filamentous shape (sometimes with extensive branching) (Fig. 1), with U-shaped, 6-shaped, or circular forms produced in tissue culture. Particles vary greatly in length (up to 14,000 nm), probably because of aggregation, but have a uniform diameter of about 80 nm. Unit length associated with peak infectivity is 790 nm for Marburg and 970 nm for Ebola viruses. Virions posses a lipid membrane envelope derived from the host cell plasma membrane. Surface projections, about 10 nm in length, are spaced at 10 nm intervals and are formed by the viral glycoprotein (GP). The nucleocapsid inside the virus envelope has a central axis (about 20 nm in diameter) surrounded by a helical nucleocapsid (about 50 nm in diameter) with cross-striations exhibiting a periodicity of about 5 nm. It is composed of the genomic RNA and the large (L) protein, nucleoprotein (NP), and virion proteins (VP) 35 and 30. There are no other visible capsomers on electron micrographs.
Physicochemical and Physical Properties
Virion Mr is 3.82 108. Buyoant density of virions is 1.14 g/cm3 as determined by centrifugation in potassium tartrate gradient. The S20w of bacilliform particles is 1.40, for longer particles it is very high. In CsCl the nucleocapsid has a buoyant density of about 1.32 g/cm3. There are no data about pH stability. The infectivity of Marburg and Ebola viruses is stable at less than 20°C, but drastically reduced within 30 minutes at 60°C. There are no data about metal cation stability. Virus infectivity is sensitive to quarternary ammonium salt, hypochlorite and phenolic disinfectants, lipid solvents, -propiolactone, formaldehyde, ultraviolet and gamma irradiation.
The particles of Marburg virus and the Ebola viruses contain seven proteins. They include the L protein that is an RNA-dependent RNA transcriptase-polymerase; the surface glycoprotein (GP) that makes up the viral spikes in the form of trimers; the nucleoprotein (NP); the matrix or membrane-associated virion protein (VP) 40; the VP35 protein that may be a transcriptase-polymerase component; the VP30 protein that may be a minor nucleoprotein; and the second matrix or membrane-associated protein VP24. The sizes of the proteins are shown in Table 1.
Virions contain a non-segmented, negative stranded, linear RNA molecule about 19 kb in size (Marburg virus 19.1 kb, Zaire Ebola virus 18.9 kb). The Mr of the genomic RNA is 4.2 106 and the genome represents about 1.1% of the total virion mass. Genomic RNA is not polyadenylated at the 3-end and there is no evidence for a 5-terminal cap structure or covalently-linked protein. Full-length nucleotide sequences of the genomes of Marburg virus (strains Musoke and Popp) and Zaire Ebola virus (strain Mayinga) have been determined.
The viral envelope is derived from the host cell membranes and is considered to have a lipid composition similar to that of the plasma membrane.
GP of filoviruses are highly glycosylated with N-linked glycans of the complex, hybrid and oligomannosidic type, and O-linked glycans of the neutral mucin type. The glycans constitute > 50% of the GP total mass. Zaire Ebola virus shows a higher level of sialylation than Marburg virus, and in certain cell lines the GP of Marburg virus completely lacks sialic acid. GP of Marburg virus is acylated and forms homotrimers. NP and VP30 of filoviruses are phosphorylated.
Genome Organization and Replication
Filovirus genomes are characterized by the gene order: 3-NP-VP35-VP40-GP-VP30-VP24-L-5 (Fig. 2). The extragenic sequences at the extreme 3- (leader) and 5- (trailer) ends of the genomes are conserved. They demonstrate a significantly high complementarity at their very ends. Genes are flanked by conserved transcriptional start and stop (polyadenylation) sites. Those sites contain a highly conserved pentamer 3-UAAUU-5. Most genes are separated by non-conserved intergenic sequences, but some genes overlap. Those overlaps are extremely short and limited to the highly conserved pentamer. There is a single gene overlap with Marburg virus and several overlaps with Zaire Ebola virus. The functional significance of these short overlaps is unclear, but transcription attenuation of the downstream gene has been postulated for other viruses of the order Mononegavirales. In addition, most genes posses relatively long 3- and 5- non-coding regions. In contrast to Marburg virus, the GP gene of Zaire Ebola virus consists of two overlapping ORFs which are fused through transcriptional editing (Fig. 2).
The replication strategy of filoviruses is not studied well. Ultrastructural studies indicate an association of viral particles with coated pits for the initiation of infection suggesting that filoviruses enter cells by endocytosis. The asialoglycoprotein receptor is discussed as a hepatocyte-specific receptor. Uncoating is presumed to occur in a manner analogous to that of other negative sense RNA viruses. Transcription and genome replication take place in the cytoplasm and follow in general the models of Paramyxoviridae and Rhabdoviridae. Transcription starts at the conserved start site and polyadenylation occurs at a run of uridine residues within the stop site. The 5-terminal non-coding sequences favor hairpin-like structures for all sgRNAs (mRNAs). Replication involves the synthesis of a full-length positive-stranded copy. During infection massive amounts of nucleocapsids accumulate intracellularly and form intracytoplasmic inclusion bodies. Virions are released via budding through plasma membranes. The expression strategy of the Zaire Ebola virus GP genes is unique and involves transcriptional editing (Fig. 2). The primary product of the unedited transcript (ORF1) yields a smaller non-structural glycoprotein sGP which is efficiently secreted from infected cells. Only RNA editing allows expression of full-length GP.
Antigenic and Genetic Properties
Virus infectivity is poorly neutralized in vitro. There is almost no antigenic cross-reactivity between the two genera. Marburg virus and Zaire Ebola virus GP genes differ by 57% and the phylogenetic analysis clearly separates both genera (Fig. 3). The cross-reactive species of the “Ebola virus-like” genus can be differentiated antigenically and genetically. All four species differ from one another by 37-41% at the nucleotide level. The genus “Marburg virus-like”, however, is antigenetically and genetically more homogenous. Comparative sequence analysis showed that two genetic lineages coexist with the recent isolate from Kenya (Ravn) differing from the others by 21-23% at the nucleotide level. Among strains of individual species of filoviruses the variation in nucleotide sequences has been shown to be extremely low, < 2% among distinct strains of the species Zaire Ebola virus. There seems to be less or even no genetic variability between isolates from different patients of single outbreaks (e.g., Gabon, Kikwit). All data indicate a remarkable degree of stability over time.
In Table 2 data on GP protein amino acid sequence homology between different Ebola and Marburg virus strains are presented. These data indicate the sharp difference between all the Ebola virus strains and the Marburg virus strain (> 68%) ans less but significant (> 30%) differences between the representatives of different species of Ebola virus.
Natural Reservoir and Transmission
The natural reservoir and history of filoviruses are still unknown. There is no connection of virus spread with any vector. The usual pattern seen with large outbreaks of disease in man begins with a focus of infection that disseminates to a number of patients. Secondary and subsequent episodes of disease occur following close contact with patients; such infections usually occur in family members or medical personnel. The major route of interhuman transmission of filoviruses requires direct contact with blood or body fluids, although droplet and aerosol infections may occur. Usage of contaminated syringes and needles are main sources for nosocomial infections.
In the laboratory, monkeys, mice, guinea pigs and hamsters have been infected experimentally.
Marburg virus was first isolated from hemorrhagic fever patients in Germany and Yugoslavia in 1967 infected by contact with tissues and blood from infected, but apparently healthy, monkeys (Cercopithecus aethiops) imported from Uganda. A second small outbreak of Marburg hemorrhagic fever occurred in Zimbabwe in 1975, and isolated episodes have occurred subsequently in Kenya in 1980 and 1987. Marburg virus mortality rates in humans are about 25%.
The first Ebola virus outbreaks were observed in northern Zaire in 1976 and in southern Sudan in 1976 and 1979. In 1994 the first case of Ebola virus disease occurred in western Africa, Cote d’Ivoire, when an ecologist was infected by examining a dead chimpanzee. In 1995 Ebola virus re-emerged in Kikwit, Zaire, with 316 cases and 245 deaths. From 1994 to 1997 three outbreaks of Ebola virus disease have been observed in Gabon. Ebola virus mortality rates in humans ranging between 50 and 90% depending on the species.
Reston Ebola virus was first isolated from Cynomolgus monkeys imported from the Philippines into the United States in 1989-1990, and from monkeys at an export facility located in the Philippines. Further isolates have been made from exported Asian monkeys in 1992 in Italy and in 1996 in Texas, USA. While pathogenic for naturally and experimentally infected monkeys, Reston Ebola virus may be less for humans. Serological investigations revealed the presence of filovirus-specific antibodies in the blood of people in many African countries such as Zaire, Sudan, Central African Republic, Gabon, Nigeria, Côte-d’Ivoire, Liberia, Cameroon and Kenya. This may indicate endemic areas of filoviruses.
The onset of the disease is sudden with fever, chills, headache, myalgia, and anorexia. This may be followed by symptoms such as abdominal pain, sore throat, nausea, vomiting, cough, arthralgia, diarrhea, and pharyngeal and conjunctival infection. Patients are dehydrated, apathetic, disoriented, and may develop a characteristic, nonpruritic, maculopapular centripetal rash associated with varying degrees of erythema and desquamate by day five or seven of the illness. Hemorrhagic manifestations develop during the peak of the illness; they are of prognostic value for the disease. Bleeding into the gastrointestinal tract is most prominent besides petechia and hemorrhages from puncture wounds and mucous membranes. Laboratory parameters are less characteristic. Mortality is high for the African members of the family and varies between 25 and 90% depending on the virus strain. Reston Ebola virus seems to possess a very low pathogenicity for humans or even be apathogenic.
The pathologic changes in fatal Marburg and Ebola hemorrhagic fever human cases include hemorrhagic diatheses into skin, mucous membranes, visceral organs and the lumen of the stomach and intestine. There is swelling of spleen, lymph nodes, kidneys, and, especially, brain. Microscopic changes include focal necroses of liver, lymphatic organs, kidneys, testes and ovaries. Most prominent are focal necroses of the liver parenchyma. Involved hepatocytes often contain large intracytoplasmic inclusion bodies, which are coincident with large amounts of viral nucleocapsids observed by electron microscopy. There is usually evidence of an interstitial pneumonia and sometimes of pancreatitis and iridocyclitis.
Filoviruses have a tropism for cells of the macrophage system, hepatocytes, adrenal cortical cells, fibroblasts, and endothelial cells. The viruses are distributed in all tissues of the body with high concentrations in the liver, kidney, spleen, and lung. Activation of the clotting cascade with hemorrhagic diathesis and fibrinolysis occurs to varying degrees depending on the virus strain.
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