MicrobiologyBytes

When it comes to nasty pathogens, Marburg virus is among the nastiest. Cousin to Ebola virus, Marburg causes fever, rash, delirium, and severe hemorrhaging, often ending in organ failure and death. It is rare in the wild, but was a central focus of weaponization by the Soviet Union, and remains a concern for terrorism experts who fear its lethal potential and resistance to treatment.

One reason that treatments have proved so elusive is because the virus is so hard to work with – hazmat suits, self-contained breathing gear, and electronically secured airlocks are all required for even the simplest of studies with live virus. But another reason is that the virion (the virus particle) is heterogeneous in shape, and that heterogeneity has confounded standard imaging techniques (X-ray crystallography, cryo-electron microscopy), which require purified, identical particles to obtain their highest resolution. Researchers have now got around that problem by using a sophisticated combination of imaging techniques that provide the first clear three-dimensional picture of the intact Marburg virion structure.

Marburg Virus Structure Revealed in Detail. (2011) PLoS Biol 9(11): e1001198. doi:10.1371/journal.pbio.1001198
and:
Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells. (2011) PLoS Biol 9(11): e1001196. doi:10.1371/journal.pbio.1001196
Several major human pathogens, including the filoviruses, paramyxoviruses, and rhabdoviruses, package their single-stranded RNA genomes within helical nucleocapsids, which bud through the plasma membrane of the infected cell to release enveloped virions. The virions are often heterogeneous in shape, which makes it difficult to study their structure and assembly mechanisms. We have applied cryo-electron tomography and sub-tomogram averaging methods to derive structures of Marburg virus, a highly pathogenic filovirus, both after release and during assembly within infected cells. The data demonstrate the potential of cryo-electron tomography methods to derive detailed structural information for intermediate steps in biological pathways within intact cells. We describe the location and arrangement of the viral proteins within the virion. We show that the N-terminal domain of the nucleoprotein contains the minimal assembly determinants for a helical nucleocapsid with variable number of proteins per turn. Lobes protruding from alternate interfaces between each nucleoprotein are formed by the C-terminal domain of the nucleoprotein, together with viral proteins VP24 and VP35. Each nucleoprotein packages six RNA bases. The nucleocapsid interacts in an unusual, flexible “Velcro-like” manner with the viral matrix protein VP40. Determination of the structures of assembly intermediates showed that the nucleocapsid has a defined orientation during transport and budding. Together the data show striking architectural homology between the nucleocapsid helix of rhabdoviruses and filoviruses, but unexpected, fundamental differences in the mechanisms by which the nucleocapsids are then assembled together with matrix proteins and initiate membrane envelopment to release infectious virions, suggesting that the viruses have evolved different solutions to these conserved assembly steps.

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Filoviruses cause lethal hemorrhagic fever in humans and nonhuman primates. The family Filoviridae includes two genera: Marburgvirus (MARV) and Ebolavirus (EBOVs). MARV was discovered in 1967 in Marburg, Germany during an outbreak in laboratory staff exposed to tissues from monkeys imported from Uganda. The Zaire virus was discovered in 1976 in Yambuku, Zaire during a 312-person outbreak associated with 90% mortality. With the exception of Reston Ebolavirus that appears to be pathogenic in nonhuman primates but not in humans and is endemic in the Philippines, all known filoviruses are pathogenic in primates including humans and are endemic in Africa. Bats are implicated as reservoirs and vectors for transmission of filoviruses in Africa. Ebolavirus sequences have been found in various bats. Bats naturally or experimentally infected with Ebolaviruses are healthy and shed virus in their feces for up to 3 weeks.

In 2002, colonies of Schreiber’s bats (Miniopterus schreibersii), sustained massive die-offs in caves in France, Spain and Portugal. This paper report the first discovery of an ebolavirus-like filovirus in bats from Europe.

Discovery of an Ebolavirus-Like Filovirus in Europe. (2011) PLoS Pathog 7(10): e1002304. doi:10.1371/journal.ppat.1002304
Filoviruses, amongst the most lethal of primate pathogens, have only been reported as natural infections in sub-Saharan Africa and the Philippines. Infections of bats with the ebolaviruses and marburgviruses do not appear to be associated with disease. Here we report identification in dead insectivorous bats of a genetically distinct filovirus, provisionally named Lloviu virus, after the site of detection, Cueva del Lloviu, in Spain.

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Ebola and Marburg virus are filoviruses that cause outbreaks of highly lethal haemorrhagic fever. Mortality rates in these diseases average more than 50%, with the highest recorded rates seen for Ebola Zaire virus (88%) and Marburg Angola virus (90%). Infection with these filoviruses produces a very high fever followed by interference with blood coagulation and vascular permeability, causing internal bleeding, bruising and skin rashes. After an asymptomatic incubation period, which can last days to weeks, symptoms of a typical filovirus infection emerge; headache, nausea, fever and malaise followed by more serious haemorrhagic symptoms and, in fatal cases, death results from multi-organ failure owing to shock.

Present treatments for filovirus infection are palliative, and consist primarily of supportive care, including hydration and pain management. There is no effective treatment or cure for these diseases. Therefore, vaccine development is crucially important as a strategy for fighting filovirus outbreaks. However, vaccine efficacy testing for Ebola virus is very difficult. There is no readily identifiable high-risk human population that can be targeted for a placebo-controlled clinical trials because disease outbreaks are unpredictable and sporadic, both geographically and temporally. Normally, clinical trials of medicines and vaccines intended for human use follow a lengthy but predictable sequence of safety and efficacy testing.

Because of its sporadic nature, the incidence of Ebola virus infection in human populations is not predictable and does not allow for adequate testing. Moreover, the immune correlates of protection from filovirus disease in humans remain unknown and therefore cannot be used to assess candidate vaccine efficacy. To facilitate the licensing of medicines when efficacy cannot be evaluated in the setting of natural infection, the U.S. Food and Drug Administration (FDA) introduced a new regulation in 2002 as an alternative licensing pathway for pharmaceutical products that target highly lethal pathogens. The FDA’s “animal rule” allows approval based on animal efficacy data. The animal rule is intended to be used as a pathway for regulatory approval only when there is no other way to licence a vaccine (Correlates of protective immunity for Ebola vaccines: implications for regulatory approval by the animal rule. 2009 Nature Reviews Microbiology 7: 393-400).

In the case of Ebola virus, the relevant animal models are non-human primates and mice. The immune correlates of Ebola virus infection consist of immunoglobulin G responses, although other factors, such as T cells, are also likely to be important in a successful immune response. Current vaccine candidates against Ebola virus include the virus glycoprotein and nucleocapsid proteins. Initial animal testing of Ebola vaccines has shown a protective effect in non-human primates and positive antibody titres in humans.

To date, no vaccines have received regulatory approval and been licensed using the FDA animal rule. This pathway does not diminish the level of regulatory contol required for vaccine approval; extensive human testing is still required to demonstrate safety and immunogenicity. The predictive relationship between animals and humans for protective efficacy is unknown, and therefore an immune correlate is used to bridge the gap between animal efficacy studies and human immunogenicity trials. It has not yet been determined what level of efficacy in animals will be required for vaccine approval, but other vaccines currently administered to the U.S. population have shown efficacies in human trials that are as low as 18%. Even this level of efficacy will provide a benefit against pathogens such as filoviruses with high mortality rates, and therefore may be acceptable against emerging natural infections or bioterrorism threats.

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