Assembly of the Marburg virus envelope.

The key player to assemble the filamentous Marburg virus particles is the matrix protein VP40 which orchestrates recruitment of nucleocapsid complexes and the viral glycoprotein GP to the budding sites at the plasma membrane. Here, VP40 induces the formation of the viral particles, determines their morphology and excludes cellular proteins from the virions. Budding takes place at filopodia in non-polarized cells and at the basolateral cell pole in polarized epithelial cells. Molecular basis of how VP40 exerts its multifunctional role in these different processes is currently under investigation. Here we summarize recent data on structure-function relationships of VP40 and GP in connection with their function in assembly. Questions concerning the complex particle assembly, budding and release remaining enigmatic are addressed. Cytoplasmic domains of viral surface proteins often serve as a connection to the viral matrix protein or as binding sites for further viral or cellular proteins. A cooperation of MARV GP and VP40 building up the viral envelope can be proposed and is discussed in more detail in this review, as the cytoplasmic domain of GP represents an obvious interaction candidate because of its localization adjacent to the VP40 layer. Interestingly, truncation of the short cytoplasmic domain of GP neither inhibited interaction with VP40 nor incorporation of GP into progeny viral particles. Based on reverse genetics we generated recombinant virions expressing a GP mutant without the cytoplasmic tail. Investigations revealed attenuation in virus growth and an obvious defect in entry. Further investigations showed that the truncation of the cytoplasmic domain of GP impaired the structural integrity of the ectodomain, whichconsequently had impact on entry steps downstream of virus binding. Our data indicated that changes in the cytoplasmic domain are relayed over the lipid membrane to alter the function of the ectodomain.

Cryo-electron tomography of Marburg virus particles and their morphogenesis within infected cells.

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.

Differentiation of filoviruses by electron microscopy.

Cultured monolayers of MA-104, Vero 76, SW-13, and DBS-FRhL-2 cells were infected with Marburg (MBG), Ebola-Sudan (EBO-S), Ebola-Zaire (EBO-Z), and Ebola-Reston (EBO-R) viruses (Filoviridae, Filovirus) and examined by electron microscopy to provide ultrastructural details of morphology and morphogenesis of these potential human pathogens. Replication of each filovirus was seen in all cell systems employed. Filoviral particles appeared to enter host cells by endocytosis. Filoviruses showed a similar progression of morphogenic events, from the appearance of nascent intracytoplasmic viral inclusions to formation of mature virions budded through plasma membranes, regardless of serotype or host cell. However, ultrastructural differences were demonstrated between MBG and other filoviruses. MBG virions recovered from culture fluids were uniformly shorter in mean unit length than EBO-S, EBO-Z, or EBO-R particles. Examination of filovirus-infected cells revealed that intermediate MBG inclusions were morphologically distinct from EBO-S, EBO-Z, and EBO-R inclusions. No structural difference of viral inclusion material was observed among EBO-S, EBO-Z, and EBO-R. Immunoelectron microscopy showed that the filoviral matrix protein (VP40) and nucleoprotein (NP) accumulated in EBO-Z inclusions, and were closely associated during viral morphogenesis. These details facilitate the efficient and definitive diagnosis of filoviral infections by electron microscopy.

Ultrastructure of Ebola virus particles in human liver.

Electron microscopy of tissues from two necropsies carried out in the Sudan on patients with Ebola virus infection identified virus particles in lung and spleen, but the main concentrations of Ebola particles were seen in liver sections. Viral precursor proteins and cores were found in functional liver cells, often aligned in membrane-bound aggregations. Complete virions, usually found only extracellularly, were mainly seen as long tubular forms, some without cores. Many tubular forms had 'enlarged heads' or 'spores' and some branched and torus forms were identified. The size and structure of the Ebola virus forms appear to be virtually indistinguishable from those of Marburg virus.

Emerging and reemerging of filoviruses.

Filoviruses are causative agents of a hemorrhagic fever in man with mortalities ranging from 22 to 88%. They are enveloped, nonsegmented negative-stranded RNA viruses and are separated into two types, Marburg and Ebola, which can be serologically, biochemically and genetically distinguished. In general, there is little genetic variability among viruses belonging to the Marburg type. The Ebola type, however, is subdivided into at least three distinct subtypes. Marburg virus was first isolated during an outbreak in Europe in 1967. Ebola virus emerged in 1976 as the causative agent of two simultaneous outbreaks in southern Sudan and northern Zaire. The reemergence of Ebola, subtype Zaire, in Kikwit 1995 caused a worldwide sensation, since it struck after a sensibilization on the danger of Ebola virus disease. Person-to-person transmission by intimate contact is the main route of infection, but transmission by droplets and small aerosols among infected individuals is discussed. The natural reservoir for filoviruses remains a mystery. Filoviruses are prime examples for emerging pathogens. Factors that may be involved in emergence are international commerce and travel, limited experience in diagnosis and case management, import of nonhuman primates, and the potential of filoviruses for rapid evolution.

[Submicroscopic characteristics of Marburg virus and its mini genome analog replication in cell cultures]. / Submikroskopicheskie osobennosti replikatsii virusa Marburg i ego mini-genomnogo analoga v kul'turakh kletok.

Marburg virus (Filoviridae) causes severe hemorrhagic fevers in humans and some lower primates with high mortality. The virus genome is formed by a single strand RNA of negative polarity, coding for seven structural proteins. We studied the ultrastructure of Marburg virus replicative cycle and replication of its minigenome RNA (coding for the terminal areas of the genome) in the presence of helper virus in VERO fibroblastoid cell culture and epithelioid MDCK cell culture. Ultrastructural parameters of Marburg virus multiplication in these cell cultures are virtually the same. The virus nucleocapsid assembly is performed on the outer side of EPR membrane and is not associated with preliminary accumulation of the precursor material. Virions form by budding on plasmalemma and are located on the entire surface in Vero cells and only on the basolateral surface of MDCK cells. Replication of minigenome analog of marburg virus is associated with impairment of the helper virus morphogenesis and formation of spherical pseudoviral particles.

[A promising method for preparative production and purification of the Marburg virus]. / Perspektivnyi metod preparativnoi narobotki i ochistki virusa Marburg.

A method for the production of Marburg virus in preparative amounts has been developed. It is based on sedimentation of the virus from blood plasma of infected guinea pigs by ultracentrifugation followed by purification in sucrose density gradient or gel chromatography on macroporous glass sorbents. The optimal terms of blood collection in infected animals were determined. Purified virus did not lose its biological activity. Concentrated virus preparations were studied by electron microscopy and polyacrylamide gel electrophoresis.

[Characterization of Marburg virus morphology].

Ebola virus (EBOV) and Marburg virus (MARV) belong to the family Filoviridae. Filoviruses cause severe filovirus hemorrhagic fever (FHF) in humans, with high case fatality rates, and represent potential agents for bioterrorism and biological weapons. It is necessary to keep surveillance of filoviruses, even though there is no report of their isolation and patients in China so far. To characterize MARV morphology, the Lake Victoria marburgvirus--Leiden was stained negatively and observed under a transmission electron microscope which is one of important detection methods for filoviruses in emergencies and bioterrorism. MARV showed pleomorphism, with filamentous, rod-shaped, cobra-like, spherical, and branch-shaped particles of uniform diameter but different lengths. Pleomorphism of negatively stained MARV is summarized in this article, so as to provide useful information for possible electron microscopic identification of filoviruses in China.

Budding of Marburgvirus is associated with filopodia.

Viruses exploit the cytoskeleton of host cells to transport their components and spread to neighbouring cells. Here we show that the actin cytoskeleton is involved in the release of Marburgvirus (MARV) particles. We found that peripherally located nucleocapsids and envelope precursors of MARV are located either at the tip or at the side of filopodial actin bundles. Importantly, viral budding was almost exclusively detected at filopodia. Inhibiting actin polymerization in MARV-infected cells significantly diminished the amount of viral particles released into the medium. This suggested that dynamic polymerization of actin in filopodia is essential for efficient release of MARV. The viral matrix protein VP40 plays a key role in the release of MARV particles and we found that the intracellular localization of recombinant VP40 and its release in form of virus-like particles were strongly influenced by overexpression or inhibition of myosin 10 and Cdc42, proteins important in filopodia formation and function. We suggest that VP40, which is capable of interacting with viral nucleocapsids, provides an interface of MARV subviral particles and filopodia. As filopodia are in close contact with neighbouring cells, usurpation of these structures may facilitate spread of MARV to adjacent cells.

Ebola and Marburg viruses: I. Some ultrastructural differences between strains when grown in Vero cells.

A strain of Marburg virus and two strains of Ebola virus grown in Vero cells were compared by electron microscopy. The outer coat of the Marburg virion appeared to be more resistant to erosion by negative staining techniques than that of the Epbola strains. Marburg virus commonly produced "torus" forms and short filaments; the Zaire strain of Ebola produced extensive branched forms and very long filaments; the Sudan strain of Ebola produced shorter, less branched structures but very many aberrant forms. The mechanism for the production of these aberrant forms is described.

Glycosylation and oligomerization of the spike protein of Marburg virus.

The oligosaccharide side chains of the glycoprotein of Marburg virus (MW 170,000) have been analyzed by determining their sensitivity to enzymatic degradation and their reactivity with lectins. It was found that they consist of N- and O-glycans. Studies employing chemical cross-linking showed that the glycoprotein is present as a homotrimer in the viral envelope.

Apoptosis induced in vitro and in vivo during infection by Ebola and Marburg viruses.

Induction of apoptosis has been documented during infection with a number of different viruses. In this study, we used transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling to investigate the effects of Ebola and Marburg viruses on apoptosis of different cell populations during in vitro and in vivo infections. Tissues from 18 filovirus-infected nonhuman primates killed in extremis were evaluated. Apoptotic lymphocytes were seen in all tissues examined. Filoviral replication occurred in cells of the mononuclear phagocyte system and other well-documented cellular targets by TEM and immunohistochemistry, but there was no evidence of replication in lymphocytes. With the exception of intracytoplasmic viral inclusions, filovirus-infected cells were morphologically normal or necrotic, but did not exhibit ultrastructural changes characteristic of apoptosis. In lymph nodes, filoviral antigen was co-localized with apoptotic lymphocytes. Examination of cell populations in lymph nodes showed increased numbers of macrophages and concomitant depletion of CD8+ T cells and plasma cells in filovirus-infected animals. This depletion was particularly striking in animals infected with the Zaire subtype of Ebola virus. In addition, apoptosis was demonstrated in vitro in lymphocytes of filovirus-infected human peripheral blood mononuclear cells by TEM. These findings suggest that lymphopenia and lymphoid depletion associated with filoviral infections result from lymphocyte apoptosis induced by a number of factors that may include release of various chemical mediators from filovirus-infected or activated cells, damage to the fibroblastic reticular cell conduit system, and possibly stimulation by a viral protein.

Ebola and Marburg viruses: II. Thier development within Vero cells and the extra-cellular formation of branched and torus forms.

The development of Marburg virus and the Sudanese and Zaire strains of Ebola virus in Vero cells as visualized by electron microscopy is described. Despite differences in timing, all three strains appear to pass through identical stages of development. Initially there is a large increase in nucleolus material, and viral precursor material arranges itself in spirals and then into tubes. The cells fill with core material, which passes to the plasmalemma, which often proliferates. Each virion passes through the plasmalemma, acquiring a coat of host material. The formation of torus forms is discussed; the branched appearance that is often seen is believed to be an aberrant form. The reasons for this view are put forward.

[Recent Lassa, Marbourg and Ebola viruses in African tropical viruses. I. Semiology--physiopathology--diagnosis--treatment (author's transl)]. / Les virus Lassa, Marbourg et Ebola, nouveaux venus en pathologie tropicale africaine. I. Sémiologie--Physio-pathologie--Diagnostic--Traitement.

Three new viruses have been identified in Africa during the present decade. They may cause sporadic cases or limited outbreaks, and they are probably endemic in areas which are still ill-defined. Severe forms of infection lead to the haemorrhagic syndrome or to hypovolemic shock, the physiopathology of which is being studied. The case-fatality ratio of severe cases is between 30 and 85 per cent. Nosocomial outbreaks have been observed, but they can be avoided if appropriate barrier nursing measures are carried out for the treatment of patients or adequate protection measures for sampling and examination of laboratory specimens. As such cases may be transferred outside the endemic zone, this implies that countries receiving travellers from Africa should have hospitals with specialized units for strict isolation and treatment of these patients.

Filovirus-induced endothelial leakage triggered by infected monocytes/macrophages.

The pathogenetic mechanisms underlying hemorrhagic fevers are not fully understood, but hemorrhage, activation of coagulation, and shock suggest vascular instability. Here, we demonstrate that Marburg virus (MBG), a filovirus causing a severe form of hemorrhagic fever in humans, replicates in human monocytes/macrophages, resulting in cytolytic infection and release of infectious virus particles. Replication also led to intracellular budding and accumulation of viral particles in vacuoles, thus providing a mechanism by which the virus may escape immune surveillance. Monocytes/macrophages were activated by MBG infection as indicated by tumor necrosis factor alpha (TNF-alpha) release. Supernatants of monocyte/macrophage cultures infected with MBG increased the permeability of cultured human endothelial cell monolayers. The increase in endothelial permeability correlated with the time course of TNF-alpha release and was inhibited by a TNF-alpha specific monoclonal antibody. Furthermore, recombinant TNF-alpha added at concentrations present in supernatants of virus-infected macrophage cultures increased endothelial permeability in the presence of 10 micron H2O2. These results indicate that TNF-alpha plays a critical role in mediating increased permeability, which was identified as a paraendothelial route shown by formation of interendothelial gaps. The combination of viral replication in endothelial cells (H.-J. Schnittler, F. Mahner, D. Drenckhahn, H.-D. Klenk, and H. Feldmann, J. Clin. Invest. 19:1301-1309, 1993) and monocytes/macrophages and the permeability-increasing effect of virus-induced cytokine release provide the first experimental data for a novel concept in the pathogenesis of viral hemorrhagic fever.

Multivesicular bodies as a platform for formation of the Marburg virus envelope.

The Marburg virus (MARV) envelope consists of a lipid membrane and two major proteins, the matrix protein VP40 and the glycoprotein GP. Both proteins use different intracellular transport pathways: GP utilizes the exocytotic pathway, while VP40 is transported through the retrograde late endosomal pathway. It is currently unknown where the proteins combine to form the viral envelope. In the present study, we identified the intracellular site where the two major envelope proteins of MARV come together as peripheral multivesicular bodies (MVBs). Upon coexpression with VP40, GP is redistributed from the trans-Golgi network into the VP40-containing MVBs. Ultrastructural analysis of MVBs suggested that they provide the platform for the formation of membrane structures that bud as virus-like particles from the cell surface. The virus-like particles contain both VP40 and GP. Single expression of GP also resulted in the release of particles, which are round or pleomorphic. Single expression of VP40 led to the release of filamentous structures that closely resemble viral particles and contain traces of endosomal marker proteins. This finding indicated a central role of VP40 in the formation of the filamentous structure of MARV particles, which is similar to the role of the related Ebola virusVP40. In MARV-infected cells, VP40 and GP are colocalized in peripheral MVBs as well. Moreover, intracellular budding of progeny virions into MVBs was frequently detected. Taken together, these results demonstrate an intracellular intersection between GP and VP40 pathways and suggest a crucial role of the late endosomal compartment for the formation of the viral envelope.

Virologic investigation of a case of suspected haemorrhagic fever.

After travelling in subSaharan Africa, an area known for sporadic cases of Marburg virus infection, a young Swedish man presented with a classical picture of severe viral haemorrhagic fever complicated by disseminated intravascular coagulation and septicaemia. Serum samples examined by electron microscopy revealed particles of a size compatible with filovirions. Indirect fluorescent antibody tests indicated transient seroconversion to Marburg virus. In lymphocyte transformation assays of cells isolated from the patient 11 months after the onset of acute disease, Marburg viral antigen was able to stimulate lymphocyte proliferation 3.9-fold; however, exhaustive attempts to isolate virus from acute phase blood cultured in vitro or in vivo from guinea pigs and monkeys failed. Data suggest that this patient may have been infected with a filovirus. This case demonstrates the difficulties that may occur in laboratory diagnosis of viral haemorrhagic fevers.