Heal The Girls: Molecular aspects of Ebola and other Filoviruses

In the last few days news organizations reported an outbreak of Ebola in West Africa, leading (as of March 24th) to a death of 59 out of 80 infected people.

The Ebola virus and its variant Marburg virus are known to be one the most lethal viruses infecting humans. Once infected patients die of hemorrhagic fever, a painful and agonizing death characterized by fever, excessive blood loss and diarrhea.

There is no effective therapy, except replacing fluids, blood, coagualnts as well as relatively generic measures such as the administration of Immunoglobulin or antiviral pharmaceuticals such as Ribavirin or S--adenosylhomocysteine (SAH) hydrolase inhibitors. Antibiotics are also given to prevent secondary infections should the patient survive. Novel treatments include the use of antisense RNA, which has been shown to treat infections in non-human primates under experimental conditions. The application of a recombinant vesicular stomatitis virus expressing the Glycoprotein from Ebola has been developed as well and might prevent death of the patient.

In order to prevent the infection from spreading into the community, patients have to be quarantined and caregivers have to wear protective clothing. The mortality is high, varying from 90% in the case of Ebola Zaire to 34% to Ebola Bundibugyo; a special case might be Ebola Reston, which did not cause any fatality among humans –although it is not clear if humans can get infected in the first place.

The following strains of Ebola virus have been identified during past epidemics:

Ebola Zaire (1976)
Ebola Sudan (1976)
Ebola Côte d'Ivoire (1994; also known as Tai Forest Virus and may only be a close relative to Ebola)
Ebola Reston (1994; causing simian hemorrhagic fever, not infectious for humans)
Ebola Bundibugyo (2007)

A relative of Ebola, Marburg Virus (closely related to Ebola but distinct from) was identified in the 1960s to be the causative agent of a small epidemic of hemorrhagic fever among animal care workers in Marburg/Germany and Yugoslavia with a fatality rate of 23-90%.

In general the first symptoms of disease include a general malaise with Influenza-like symptoms, including fever/chills, chest pain, and phryngitis. If the central nervous is affected symptoms include severe headache, depression, confusion, fatigue, and coma. The most visible symptoms include hemorrhagic symptoms - such as bleeding at injection sites, and hematomas. Death generally occurs because of low blood pressure, tissue necrosis and multiple organ dysfunction.

Ebola and Marburg virus’ belong to the order of the Mononegavirales -an order which includes other viruses of interest, Hendra and Nipah Virus, both which cause serious diseases in their own right- and the family of Filoviridiae.

Organisation of the Filovirus genome

As such, the viruses have a negative strand RNA genome with a length of approximately 19kb, encoding for seven genes (NP, VP35,VP40,(s)GP, VP30, VP24 and L) each gene is flanked by a Non Translated (NTS) 3’ leader sequence and a 5’ trailer sequence (see figure), a feature shared with Nipah and Hendra viruses. Furthermore a non-coding region of varying length separates most genes. Five of the proteins encoded are shared with other negative ssRNA viruses (including the RNA dependent RNA Polymerase or L-protein and the Glycoprotein (GP)), whilst the Viral Proteins (VP) 35 and 40 are unique to Filoviruses.

Ebola viruses, similar to other Filoviridiae, infect not only human and non-human primates but other animals such as pigs and some species of fruit bats as well, in addition to a wide variety of cell lines. Cell types susceptible to Filovirus infection include but not limited to adrenal cortical cell, hepatocytes, endothelial cells, fibroblasts, dendritic cells, monocytes, and macrophages, thus explaining the wide range of symptoms in infected individuals.

Following exposure to both Ebola and Marburg virus, early targets of the virus include cells constituting the immune system -macrophages, dendritic cells and monocytes. This allows the virus to be spread to other parts of the body via the lymphatic and blood system, reaching the liver and the intestine. In addition, non-infected lymphocytes are depleted by probably via the induction of bystander apoptosis rather than infection with nascent virus.

Functions of the proteins

As mentioned above the L gene encodes the RNA dependent RNA Polymerase that is required for the conversion of the negative ssRNA into the positive strand RNA which serves as a template for negative ssRNA (to be incorporated into the genome of newly synthesized viral particles) as well as the synthesis of seven monocistronic mRNA species.

Simplified illustration of the Filovirus replication cycle

The glycoprotein is incorporated into the membrane of viral particles and –upon infection- binds to the receptor of the host cell, whilst the function of the soluble Glycoprotein (which is secreted and not incorpor-ated into the nucleocapsid and unique to Ebola virus) is unknown.

The Nucleocapsid (NP) and VP 35/24 proteins are required for the assembly of the viral particle whereas VP 30 is required for viral budding – the release of the virus particle from the host cell. In addition of its function in the assembly of the viral particle the Nucleocapsid protein might also be involved in the transport of the pre-assembled particle to the surface of the infected cell along the microtubuli. Prior to its incorporation in the virus particle the Glycoprotein is modified within the Golgi.

Because of the mortality of Ebola and Ebola related viruses, outbreaks are self limiting and relatively small in terms of numbers of people infected. The virus is also mostly transmitted by close contact with infected patients in addition to poor sanitary conditions and contaminated water and food. The virus is believed to have originally been limited to infect primates and fruit bats – although the natural host has not been identified with absolute certainty. It crossed the species barrier only when humans started to explore those areas. Ebola and Ebola-related diseases are considered to be zoonotic –of animal origin. In the case of Ebola, larger epidemics have been avoided so far, probably thanks to the high mortality this virus exhibits.

Interactions between viral proteins and the host cell

In recent years detailed analysis has revealed a number of cellular proteins which are interacting with different viral proteins during the infectious cycle of Ebola virus.

Most notably this work has identified not only the cellular receptors required for viral entry but also proteins required for the release of the viral genome into the cytoplasm of the host cell. As it turns out both the fusion of Filoviruses and the release of the genome into the cytoplasm closely resemble the mechanism found in Influenza virus and other pathogens. It should be noted that there is no bona fide receptor for Ebola or Marburg virus, but it seems that some might function as co-receptor or that the virus can bind different receptors. Viral entry itself is mediated by lipid raft dependent mechanisms and macropinocytes, a specialized subtype of endocytosis. Following entry into the cytoplasm, the viral genome is released in a Cathepsin B and L dependent process similar to the release of the Influenza virus genome, although this varies between cell lines (in Vero cells, Cathepsin L is required but is dispensable in human dendritic cells).

Studies using different cell lines confirmed the presence of a clear cytopathic effect (CPE) , although with a different severity which depends not only on the virus but also on the cell line used. Interestingly primary human cells -macrovascular or microvascular endothelial cells as well as macrophages and monocytes derived from normal peripheral blood -infected with Ebola do not exhibit any CPE. Cell death of other cell types infected -such as hepatocytes- seems to be non-apototic although Ebola virus does not antagonize apoptotic signaling pathways.

More recent results enabled to visualize the formation of nascent viral particles in distinct inclusion bodies by live cell microscopy. In theory it might be possible now to almost observe viral infection "live as it happens", thus leading to new insights into the biology and pathogenesis of diseases caused by Filoviruses.

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Molecular aspects of Ebola and other Filoviruses

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