Filoviruses

Anthony Sanchez, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Filoviruses are a group of mysterious and highly pathogenic RNA viruses that are primarily associated with equatorial regions of Africa. Filoviruses are genetically related to the more widely known mumps, measles and rabies viruses.

Secondary article
Article Contents
. Classification . Structure . Genetics and Replication . Clinical Features . Diagnosis

Classification
Filoviruses are currently classied in the order Mononegavirales, the family Filoviridae, and the genera Marburg-like viruses and Ebola-like viruses. Although Marburg and Ebola viruses are structurally similar and cause comparable diseases in man, these two groups of loviruses are nonetheless quite distinct in their biology. Marburg virus gets its name from a city in Germany, one of the sites where the rst identied episode of lovirus disease in humans took place in 1967. Ebola virus was rst identied in 1976 and was named after a river located in the northern region of the Democratic Republic of the Congo (the former Zaire), one of the areas of the original outbreak. The family Filoviridae is evolutionarily quite divergent, having one species of Marburg virus and four species of Ebola virus (Zaire, Sudan, Reston and Co te dIvoire). The genetic variation in the family is illustrated in Figure 1, which shows an evolutionary tree of the family based on phylogenetic analysis of their glycoprotein genes.

. Epidemiology and Natural History

structural glycoprotein (GP), VP40 protein, and VP24 protein. The GP forms trimers and facilitates the attachment of virions to host cells and the introduction of the ribonucleoprotein complex into the cytoplasm. The role of the VP40 (matrix) protein is to promote the union of nucleocapsids to the outer membrane and budding of the virion. No role for the VP24 protein has been identied.

Genetics and Replication

As with all members of the order Mononegavirales, loviruses contain a single, negative-sense RNA as their
Marburg

Structure
The virions of loviruses (L. lum thread) are characterized by an unusual rod-shaped morphology (Figure 2) that can appear very pleomorphic, especially when produced in tissue culture. Virions are approximately 80 nm in diameter, and the smallest lengths of infectious particles are roughly 800 nm for Marburg virus and 1000 nm for Ebola viruses. Pleomorphic forms can appear circular (doughnutshaped), U-shaped, and 6-shaped; long lamentous forms can also be found that reach lengths up to 14 mm. Virions contain seven structural proteins, each of which is produced from a separate gene. Figure 3 illustrates a lovirus virion with the components of the particle and their locations identied. Virions are composed of a nucleocapsid, or ribonucleoprotein complex, that is surrounded by a lipid bilayer envelope derived from the host plasma (outer) membrane from which the virus buds and is released from the cell. The nucleocapsid is formed from a strong association of the nucleoprotein and VP30 protein with the genomic ribonucleic acid (RNA), along with the weaker bound polymerase and VP35 protein complex, which provides enzymatic activity in viral RNA synthesis. Membrane-associated proteins include the
Zaire Ebola Cte dIvoire Sudan Reston
Figure 1 Evolutionary tree of the family Filoviridae based on the coding regions of glycoprotein gene sequences. Branch lengths correspond to nucleotide differences between virus isolates for which transversions have been weighted 4:1 over transitions.

Figure 2 Scanning electron micrograph of a cultured monkey kidney cell infected with a Zaire species of Ebola virus. Virions are seen budding from the entire surface of the cultured cell. Courtesy of C S Goldsmith, Centers for Disease Control and Prevention, Atlanta.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

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Glycoprotein (GP; trimer) Polymerase (L) and VP35 Nucleoprotein (NP) VP40 Membrane VP24 Genomic RNA VP30

Figure 3 Representation of a filovirus virion depicting the locations and associations of the structural proteins with the genomic RNA or outer membrane. The ribonucleoprotein complex (nucleocapsid) is surrounded by a lipid bilayer derived from the outer cell membrane. VPn, virion protein of nkDa molecular size.

genome. Replication of the virus is similar to that of other families in the order (Figure 4). From genetic analyses of a 1976 isolate of the Zaire species of Ebola virus and 1967 and 1980 isolates of Marburg virus, it has been determined that seven genes are contained in RNA genomes that are approximately 19 000 bases in length. Owing to the negative-strand nature of these viruses, the rst event following release of the nucleocapsid into the host cell cytoplasm is transcription of the genome. Positive-sense, polyadenylated messenger RNA (mRNA) molecules are copied from the genome by the viral RNA polymerases (encapsidated RNA acts as template). They are then translated by cellular ribosomes, and eventually a build-up of viral proteins leads to a switch to replication. This switch leads to synthesis of plus-sense nucleocapsids, which serve as templates for production of large numbers of negativesense nucleocapsids. Eventually, an equilibrium between transcription and replication develops that leads to the assembly of virions when sucient levels of nucleocapsids and envelope-associated proteins are reached. A major
Non-structural glycoprotein (SGP; Ebola viruses only) Release Structural proteins Budding Translation (+) Transcription Entry () Replication Binding = mRNA = Nucleocapsids (+) = GP, VP40 and VP24 = NP, VP30, L and VP35 ()

dierence between Ebola viruses and Marburg virus is seen in the expression of the glycoprotein gene. Ebola viruses are unusual in that, in addition to a structural GP, they produce a nonstructural protein, SGP, which is expressed as the primary gene product. SGP forms homodimers that are secreted from infected cells.

Clinical Features
Filoviruses cause a severe haemorrhagic fever disease in human and/or nonhuman primates, and of all the haemorrhagic fever viruses, loviruses have the highest case-fatality rates (3090%). An incubation period of 410 days is followed by a sudden onset of inuenza-like signs or symptoms (e.g. fever, chills, headache, myalgia, anorexia, malaise). Deterioration follows and patients can exhibit pharyngitis, nausea, vomiting and neurological symptoms (i.e. tinnitus, hearing loss, sudden blindness). In severe cases, haemorrhagic manifestations can develop, including bloody diarrhoea, blood clotting dysfunctions, leakage of blood into tissues from capillaries (rash or petechiae) and uncontrolled bleeding from venepuncture sites. Such cases are frequently fatal as a result of haemorrhaging and a shock syndrome, usually occurring 69 days after onset of disease. Laboratory ndings reveal an early lymphopenia, followed by a neutrophilia, marked thrombocytopenia (50100 109 cells per litre), abnormal platelet aggregation, elevated serum enzyme levels (aspartate aminotransferase exceeds alanine aminotransferase), hyperproteinanaemia, and proteinuria. In the later stage of disease, there may be complications caused by opportunistic infections. Individuals who survive infections require 5 or more weeks to recover, but no permanent damage to the body occurs. Tissue specimens obtained from outbreaks and experimental infection of monkeys and guinea-pigs have been used to study the pathology of lovirus infections. Examination has revealed that lovirus replication is very pronounced in liver, spleen, lymph nodes and lungs. Severe necrosis is especially evident in the liver and spleen late in the disease. Little or no inammation is evident in fatal infections, but there appears to be a prominent involve-

Figure 4 Representation of a cell showing the replication of a filovirus in the cytoplasm of a cell. Depicted from left to right are the binding and entry of an infecting virion, virus protein expression and RNA replication, and the assembly and release of a new virion. The 1 and 2 signs indicate the sense of the RNA in nucleocapsids or messenger RNA. Ebola viruses differ from Marburg virus in that the former produce a nonstructural, secreted glycoprotein (SGP) in addition to the seven structural proteins.

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ment of the mononuclear phagocytic and reticuloendothelial systems. Infected macrophages are easily detected and may serve as an important target cell early in the disease, and may facilitate the spread of the virus throughout the body. Disseminated intravascular coagulation has been noted and may be an important process in the pathogenesis of lovirus haemorrhagic fevers and the shock syndrome that leads to death. Increased endothelial leakage is another component of the shock syndrome, but the mechanisms behind the leakage are not dened; in vitro studies have shown that production of the cytokine tumour necrosis factor a may be involved in increased permeability. One cannot overlook, however, the cytopathic eects that lovirus infection of the reticuloendothelial system have on the vascular instability. No antiviral drug or vaccine is currently available for use in humans to protect against lovirus infections.

Immunohistochemical staining of formalin-xed tissues has been used to detect Ebola virus antigen during recent outbreaks, and is very useful when specimens cannot be preserved by refrigeration or freezing; large amounts of antigen can be seen in skin snips from fatal cases. The use of reverse transcriptase polymerase chain reaction (RT-PCR) assays to detect virus RNA in fresh samples of body uids or tissues is also possible and is very sensitive; however, these assays require more expertise and safeguards to ensure accurate and reproducible results. RT-PCR products also provide sequence information for epidemiological and evolutionary investigations.

Epidemiology and Natural History

Outbreaks of lovirus infections in humans and nonhuman primates are relatively rare events, and the spread of disease is inecient. Transmission of lovirus disease generally requires physical contact with an infected individual or infectious substance, but can also occur through inhalation of or ocular contact with droplets generated from the body uids of an infected patient. However, aerosol spread similar to that of inuenza or cold viruses has not been demonstrated for loviruses. Outbreaks can thus be easily handled by limiting the contact of infected persons with the public and supporting health care workers. This is best accomplished by isolating the patient, instituting barrier nursing techniques, and immediately disinfecting contaminated materials. The natural host and maintenance strategy for loviruses is unknown, which is unusual for a family of viruses that contains prominent human pathogens. It is believed that loviruses are zoonotic (i.e. animal-borne), based on certain biological evidence and the natural hosts of similar viruses. Following the 1976 Ebola virus outbreaks in Sudan and the former Zaire, there was some speculation that bats may be the reservoir for loviruses, but attempts at identifying lovirus infections in these or any other animal have not been successful. Recently, it was demonstrated that fruit and insectivorous bats could support replication and circulation of large amounts of Ebola (Zaire) virus in their blood without any apparent illness. Nevertheless, eorts to detect loviruses in bats and other animals trapped at sites of outbreaks have so far been unsuccessful, and clues as to the ecology of loviruses have not been forthcoming. In general, loviruses are geographically linked to the central region of the African continent, except for the Reston species of Ebola virus, which has been associated with infections of monkeys originating in the Philippines but is nonpathogenic in humans. Due to the extreme pathogenicity of loviruses in nonhuman primates, it is likely that, as with humans, they are merely victims of lovirus infections. Presumably, these infections occur as a
3

Diagnosis
Diagnosis of acute infections is most easily performed using an antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect lovirus antigen (proteins) in the blood. An ELISA system can also be used to detect early humoral responses (immunoglobulin M, IgM) to lovirus infections, but oftentimes humoral responses are weak or totally absent in severe cases. IgM or IgG ELISAs can be used to diagnose convalescent or recovered cases; IgM titres can wane over time. Alternative serological assays include indirect uorescent assays (IFAs), Western blot assays and radioimmunoprecipitation (RIP) assays. These tests are generally less desirable than ELISA formats, owing to nonspecic reactions of the IFA test and the inherent diculties associated with Western blot and RIP assays, especially when testing large numbers of specimens. The IFA test is useful, however, to check impression smears of tissues for antigen quickly. Filoviruses can be readily isolated/grown from fresh tissue and body uid samples when cultured in monkey kidney cell lines, such as Vero E6 or MA104 cells; the more pathogenic strains can exhibit extensive cytopathic eects within a week. Virus isolation is relatively simple, but working with and amplifying infectious virions poses a biosafety risk, and isolation attempts should only be performed in a maximum-containment facility known as a biosafety level 4 (BL4) laboratory. Growth of virus is usually monitored and conrmed by IFA, ELISA or electron microscopy (EM). Direct visualization of virus particles by EM is very useful when examining body uids if large numbers of virions are present, but this type of testing requires greater technical training and dedicated facilities, and is more time-consuming when large numbers of samples are being processed. EM can also be used to detect the characteristic lovirus morphology in tissues.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net

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natural matter of course, and spread of the virus within their species or to other animals may serve to increase the chance of transmission to humans. It is possible that such infections in wild animals may lead to adaptive mutations that are prerequisites for severe disease in humans. The search for the natural host is complicated by the fact that outbreaks of lovirus disease are rare and usually originate from remote areas, and the identication of the host will probably result from a very concerted eort or some fortuitous discovery. Identication of the natural host and characterization of the mechanisms of maintenance in the wild will be important to studies of the evolution and diversity of loviruses, as well as epidemiological studies of risk factors that lead to outbreaks. Until then, the natural

history of these pathogenic agents will continue to mystify the scientic community.

Further Reading
Feldmann H, Sanchez A and Klenk H-D (1998) Filoviruses. In: Collier L, Balows A and Sussman M (eds) Topley & Wilsons Microbiology and Microbial Infections, 9th edn, vol. 1, pp. 651664. London: Arnold. Murphy FA, Fauquet CM, Bishop DHL et al. (eds) (1995) Virus Taxonomy. Sixth report of the International Committee on Taxonomy of Viruses. Archives of Virology. Supplementum 10. Peters CJ, Sanchez A, Rollin PE, Ksiazek TG and Murphy FA (1996) Filoviridae: Marburg and Ebola viruses. In: Fields BN, Knipe DM, Howley PM et al. (eds) Fields Virology, 3rd edn, pp.11611176. Philadelphia, PA: Lippincott-Raven.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net