Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity.

To study the mechanisms underlying the high pathogenicity of Ebola virus, we have established a system that allows the recovery of infectious virus from cloned cDNA and thus permits genetic manipulation. We created a mutant in which the editing site of the gene encoding envelope glycoprotein (GP) was eliminated. This mutant no longer expressed the nonstructural glycoprotein sGP. Synthesis of GP increased, but most of it accumulated in the endoplasmic reticulum as immature precursor. The mutant was significantly more cytotoxic than wild-type virus, indicating that cytotoxicity caused by GP is down-regulated by the virus through transcriptional RNA editing and expression of sGP.

Isospora hypoleucae sp. n. (Apicomplexa: Eimeriidae), a new coccidian parasite found in the Pied Flycatcher (Ficedula hypoleuca).

A new Coccidia species is reported from the natural population of Pied Flycatcher (Ficedula hypoleuca) in northern Germany. Sporulated oocysts were found in faeces from 6 of 8 sampled adults. The spherical oocysts of the new Isospora species have a brownish, smooth, bi-layered wall. Average size of sporulated oocysts was 19.4 x 19.3 microm (17.5-22.8 microm x 17.5-22.8 microm ) with a shape index (length/width) of 1.0. The sporulated oocysts have no micropyle or residuum, but enclose several small polar granules that often cluster into 2-3 dumbbell-shaped formations. Sporocysts are slightly elongated, rounded at the end opposite the Stieda body, 15.3 microm x 9.2 microm in size (13.8-16.1 microm x 8.5-10.3 microm ), and have a shape index of 1.7 (1.6-1.8). The Stieda body has a prominent knob-like cap, whereas the substieda body is absent. Sporocysts contain a small compact sporocyst residuum and 4 sporozoites. COI haplotypes identical to those isolated from faecal oocysts were PCR amplified from the blood of 13-day-old nestlings, suggesting that the newly described species has extra-intestinal stages in blood. This represents the first description of a new avian Isospora species supported by molecular sequence data from the same oocysts that are described morphologically.

Structural characterization and membrane binding properties of the matrix protein VP40 of Ebola virus.

The matrix protein VP40 of Ebola virus is believed to play a central role in viral assembly as it targets the plasma membrane of infected cells and subsequently forms a tightly packed layer on the inner side of the viral envelope. Expression of VP40 in Escherichia coli and subsequent proteolysis yielded two structural variants differing by a C-terminal truncation 114 amino acid residues long. As indicated by chemical cross-linking studies and electron microscopy, the larger polypeptide was present in a monomeric form, whereas the truncated one formed hexamers. When analyzed for their in vitro binding properties, both constructs showed that only monomeric VP40 efficiently associated with membranes containing negatively charged lipids. Membrane association of truncated, hexameric VP40 was inefficient, indicating a membrane-recognition role for the C-terminal part. Based on these observations we propose that assembly of Ebola virus involves the formation of VP40 hexamers that is mediated by the N-terminal part of the polypeptide.

Parasites of Chaffinch (Fringilla coelebs) population. Part I. Coccidia (Protozoa, Apicomplexa).

Coccidia infection in Chaffinch population in Mazurian Lakeland was studied. Birds were caught in mist nets from June to September and droppings were collected after defecation. Prevalence of infection was high -80% of Chaffinches excreted oocysts of Isospora sp. Intensity of oocysts' production varied depending on the time of the day and therefore coccidia prevalence in Chaffinch should be best detectable in birds caught after midday.

Filoviruses: Interactions with the host cell.

The highly pathogenic filoviruses, Marburg and Ebola virus, are difficult to handle and knowledge of the interactions between filoviruses and their host cells remained enigmatic for many years. Two developments were crucial for the presented advances in our understanding of the cell biology of filoviruses, which is still fragmentary. On the one hand, the number of high containment laboratories increased where handling of the highly pathogenic filoviruses is possible. On the other hand, molecular biological tools have been developed that allow investigation of certain aspects of filoviral replication under normal laboratory conditions which considerably accelerated research on filoviruses. This review describes advances in understanding the interactions between host cells and filoviruses during viral attachment, entry, transcription, assembly and budding.

Crystallization and preliminary X-ray analysis of the matrix protein from Ebola virus.

The matrix protein from Ebola virus is a membrane-associated molecule that plays a role in viral budding. Despite its functional similarity to other viral matrix proteins, it displays no sequence similarity and hence may have a distinct fold. X-ray diffraction quality crystals of the Ebola VP40 matrix protein were grown by the hanging-drop vapour-diffusion method. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 64.4, b = 91.1, c = 47.9 A, beta = 96.3 degrees. A data set to 1.9 A resolution has been collected using synchrotron radiation. The unit cell contains one molecule of molecular weight 35 kDa per asymmetric unit, with a corresponding volume solvent content of 35%.

Crystal structure of the matrix protein VP40 from Ebola virus.

Ebola virus maturation occurs at the plasma membrane of infected cells and involves the clustering of the viral matrix protein VP40 at the assembly site as well as its interaction with the lipid bilayer. Here we report the X-ray crystal structure of VP40 from Ebola virus at 2.0 A resolution. The crystal structure reveals that Ebola virus VP40 is topologically distinct from all other known viral matrix proteins, consisting of two domains with unique folds, connected by a flexible linker. The C-terminal domain, which is absolutely required for membrane binding, contains large hydrophobic patches that may be involved in the interaction with lipid bilayers. Likewise, a highly basic region is shared between the two domains. The crystal structure reveals how the molecule may be able to switch from a monomeric conformation to a hexameric form, as observed in vitro. Its implications for the assembly process are discussed.

Characterization of the L gene and 5' trailer region of Ebola virus.

The nucleotide sequences of the L gene and 5' trailer region of Ebola virus strain Mayinga (subtype Zaire) have been determined, thus completing the sequence of the Ebola virus genome. The putative transcription start signal of the L gene was identical to the determined 5' terminus of the L mRNA (5' GAGGAAGAUUAA) and showed a high degree of similarity to the corresponding regions of other Ebola virus genes. The 3' end of the L mRNA terminated with 5' AUUAUAAAAAA, a sequence which is distinct from the proposed transcription termination signals of other genes. The 5' trailer sequence of the Ebola virus genomic RNA consisted of 676 nt and revealed a self-complementary sequence at the extreme end which may play an important role in virus replication. The L gene contained a single ORF encoding a polypeptide of 2212 aa. The deduced amino acid sequence showed identities of about 73 and 44% to the L proteins of Ebola virus strain Maleo (subtype Sudan) and Marburg virus, respectively. Sequence comparison studies of the Ebola virus L proteins with several corresponding proteins of other non-segmented, negative-strand RNA viruses, including Marburg viruses, confirmed a close relationship between filoviruses and members of the Paramyxovirinae. The presence of several conserved linear domains commonly found within L proteins of other members of the order Mononegavirales identified this protein as the RNA-dependent RNA polymerase of Ebola virus.

Proteolytic processing of Marburg virus glycoprotein.

Processing of the transmembrane glycoprotein (GP) of Marburg virus involved the conversion of an endo H-sensitive, ER-specific form into an endo H-resistant, Golgi-specific precursor that was cleaved into GP(1) and GP(2). Cleavage was mediated by furin or another subtilisin-like endoprotease with similar substrate specificity as indicated by mutational analysis of the cleavage site and inhibition using peptidyl chloromethylketones. Mature GP consisted of disulfide-linked GP(1) and GP(2) subunits.