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The lectins DC-SIGN and DC-SIGNR can augment viral infection; however, the range of pathogens interacting with these attachment factors is incompletely defined. Here we show that DC-SIGN and DC-SIGNR enhance infection mediated by the glycoprotein (GP) of Marburg virus (MARV) and the S protein of severe acute respiratory syndrome coronavirus and might promote viral dissemination. SIGNR1, a murine DC-SIGN homologue, also enhanced infection driven by MARV and Ebola virus GP and could be targeted to assess the role of attachment factors in filovirus infection in vivo.

We have conducted an international quality assurance study of filovirus, Lassa virus, and orthopox virus PCR with 24 participants. Of the participating laboratories, 45.8 and 66.7% detected virus in all plasma samples, which contained ≥5,000 and ≥100,000 copies per ml, respectively. Sensitivity levels were not significantly different between viruses. False-negative results were attributable to a lack of sensitivity.

As the recent SARS epidemic has shown, viruses are able to migrate with remarkably high speed, endangering countries around the globe within hours. Since viruses are obligate parasites, their migration speed is dependent on the mobility of the respective host. Several examples of pathogenic viruses with different patterns of migration will be discussed.

Several papers have been published on the genomic sequence and phylogeny of the severe acute respiratory syndrome (SARS) coronavirus ([1][1]–[4][2]). We have determined the nucleotide sequence of the SARS coronavirus FRA isolate (accession number AY310120) and found the overall genome organization

In this study, release of abundant amounts of the Ebola virus (EBOV) surface glycoprotein GP in a soluble form from virus-infected cells was investigated. We demonstrate that the mechanism responsible for the release of GP is ectodomain shedding mediated by cellular sheddases. Proteolytic cleavage taking place at amino-acid position D637 removes the transmembrane anchor and liberates complexes consisting of GP1 and truncated GP2 (GP(2delta)) subunits from the cell surface. We show that tumor necrosis factor alpha-converting enzyme (TACE), a member of the ADAM family of zinc-dependent metalloproteases, is involved in EBOV GP shedding. This finding shows for the first time that virus-encoded surface glycoproteins are substrates for ADAMs. Furthermore, we provide evidence that shed GP is present in significant amounts in the blood of virus-infected animals and that it may play an important role in the pathogenesis of infection by efficiently blocking the activity of virus-neutralizing antibodies.

Transcription of Ebola virus (EBOV)-specific mRNA is driven by the nucleocapsid proteins NP, VP35, and L. This process is further dependent on VP30, an essential EBOV-specific transcription factor. The present study addresses the self-assembly of VP30 and the functional significance of this process for viral transcription and propagation. Essential for oligomerization of VP30 is a region spanning amino acids 94–112. Within this region a cluster of four leucine residues is of critical importance. Mutation of only one of these leucine residues resulted in oligomerization-deficient VP30 molecules that were no longer able to support EBOV-specific transcription. The essential role of homo-oligomerization for the function of VP30 was further corroborated by the finding that mixed VP30 oligomers consisting of VP30 and transcriptionally inactive VP30 mutants were impaired in their ability to support EBOV transcription. The dominant negative effect of these VP30 mutants was dependent on their ability to bind to VP30. The oligomerization of VP30 could be dose dependently inhibited by a 25-mer peptide (E30pep-wt) derived from the presumed oligomerization domain (IC50,1 μm). A control peptide (E30pep-3LA), in which three leucines were changed to alanine, had no inhibitory effect. Thus, E30pep-wt seemed to bind efficiently to VP30 and consequently blocked the oligomerization of the protein. When E30pep-wt was transfected into EBOV-infected cells, the peptide inhibited viral replication suggesting that inhibition of VP30 oligomerization represents a target for EBOV antiviral drugs.

BACKGROUND: The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. METHODS: Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. RESULTS: A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. CONCLUSIONS: The novel coronavirus might have a role in causing SARS.

Localization of VP40 in Marburg virus (MBGV)-infected cells was studied by using immunofluorescence and immunoelectron microscopic analysis. VP40 was detected in association with nucleocapsid structures, present in viral inclusions and at sites of virus budding. Additionally, VP40 was identified in the foci of virus-induced membrane proliferation and in intracellular membrane clusters which had the appearance of multivesicular bodies (MVBs). VP40-containing MVBs were free of nucleocapsids. When analyzed by immunogold labeling, the concentration of VP40 in MVBs was six times higher than in nucleocapsid structures. Biochemical studies showed that recombinant VP40 represented a peripheral membrane protein that was stably associated with membranes by hydrophobic interaction. Recombinant VP40 was also found in association with membranes of MVBs and in filopodia- or lamellipodia-like protrusions at the cell surface. Antibodies against marker proteins of various cellular compartments showed that VP40-positive membranes contained Lamp-1 and the transferrin receptor, confirming that they belong to the late endosomal compartment. VP40-positive membranes were also associated with actin. Western blot analysis of purified MBGV structural proteins demonstrated trace amounts of actin, Lamp-1, and Rab11 (markers of recycling endosomes), while markers for other cellular compartments were absent. Our data indicate that MBGV VP40 was able to interact with membranes of late endosomes in the course of viral infection. This capability was independent of other MBGV proteins.

The family of Filoviridae comprises Marburg and Ebola virus which both cause severe life-threatening diseases characterized by high fever, rash, thrombocytopenia and hemorrhagic diathesis. The pathogenesis of the syndrome is not completely understood; probably the dynamic replication of filoviruses in the infected host leads to an uncoordinated immune response. Detailed understanding of the basic mechanisms of filoviral assembly and interaction with host cells is key to identify targets of antiviral intervention. The first sign of filovirus replication that can be detected microscopically in the infected cell is the formation of inclusions in the perinuclear region. Inclusions contain all filoviral nucleocapsid proteins (NP, VP35, VP30, VP24, and L) but also the matrix protein VP40 and a number of cellular proteins. Viral nucleocapsids are formed within the inclusions by specific interactions among the viral proteins. Mature nucleocapsids are transported across the cytoplasm to the plasma membrane with the help of the actin cytoskeleton. In the cell periphery nucleocapsids are associated with the matrix protein and channeled into filopodia, the site of filoviral release. Nucleocapsids inside filopodia are cotransported together with the unconventional motor protein myosin 10. Transport of nucleocapsids and release of viral particles is supported by the cellular ESCRT machinery.

A 47-year-old man sustained a 31% TBSA burn injury. In spite of early escharectomy and mesh-graft-transplantation the patient suffered a septicaemic phase in the first week, which was treated by a specific antibiotic. Five weeks after the burn injury a cervical spondylodiscitis was diagnosed. Immediate wound debridement, ventral and dorsal spondylodesis with a tricortical bone-graft from the left iliac crest and titanium plates and specific antibiotic therapy led to the stabilization and healing of the cervical spinal column. The spondylodiscitis was microbiologically proved to be hematogenous after spread of Staphylococcus aureus from the blood in the early septicaemic phase. Swab culture from the burn surface wound, infected vertebrae and blood during the septicaemic phase revealed coagulase positive S. aureus. The aetiology, predisposing factors and management of this rare, but recognized, complication of major burns are discussed. Case features of this patient are compared with the single site's reported case of hematogenous cervical spondylodiscitis after severe burn injury.

Expression of glycoproteins has been carried out successfully using recombinant vaccinia virus vectors. Especially attractive is the use of recombinant vaccinia viruses which express the DNA-dependent RNA polymerase of the phage T7 (T7-polymerase). The T7-polynerase drives the transcription of plasmid-based genes under the control of the T7 RNA polymerase promoter transfected into the infected cell. Comparison of two different recombinant vaccinia viruses, vTF7-3 and MVA-T7, revealed that post-translational processing of Marburg virus surface glycoprotein (GP) is impaired in the MVA-T7 but not in the vTF7-3 system. Influenza virus hemagglutinin, however, was transported and processed like the authentic protein in both systems. It is shown that transport of GP in the MVA-T7 system is not completely blocked, but the vast majority of molecules remained Endo H-sensitive. Only trace amounts evaded the endoplasmatic reticulum and reached the plasma membrane. Thus, the adverse effects of MVA-T7 on the processing of recombinant glycoproteins cannot be predicted, and correct processing has to be investigated for every expressed 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.

Influenza is a severe, febrile disease of the airways that occurs epidemically in the winter months. It is caused by influenza viruses. In cycles of about 30 years, these seasonal epidemics progress to pandemics with high numbers of infections and deaths worldwide. In order to prevent just this situation, so-called pandemic plans have been developed worldwide. Besides vaccination, general hygienic precautions and antiviral therapies are the most important measures in the management of influenza. We can only wait and see whether or not the newly appeared H1N1 variant of the influenza virus A (Mexican flu, swine flu) will actually turn out to be a new worldwide pandemic.

The members of the family Filoviridae , Marburg virus (MBGV) and Ebola virus (EBOV), are very similar in terms of morphology, genome organization, and protein composition. To compare the replication and transcription strategies of both viruses, an artificial replication system based on the vaccinia virus T7 expression system was established for EBOV. Specific transcription and replication of an artificial monocistronic minireplicon was demonstrated by reporter gene expression and detection of the transcribed and replicated RNA species. As it was shown previously for MBGV, three of the four EBOV nucleocapsid proteins, NP, VP35, and L, were essential and sufficient for replication. In contrast to MBGV, EBOV-specific transcription was dependent on the presence of the fourth nucleocapsid protein, VP30. When EBOV VP30 was replaced by MBGV VP30, EBOV-specific transcription was observed but with lower efficiency. Exchange of NP, VP35, and L between the two replication systems did not lead to detectable reporter gene expression. It was further observed that neither MBGV nor EBOV were able to replicate the heterologous minigenomes. A chimeric minigenome, however, containing the EBOV leader and the MBGV trailer was encapsidated, replicated, transcribed, and packaged by both viruses.

The term "the dirty dozen" refers to the 12 biological agents such as, for example, the anthrax bacillus or botulinum toxin that could be used as biological weapons. Even if their military use is considered unlikely physicians should be familiar with the problematic because these pathogens can also occur naturally or, respectively, cause disease as the consequence of a laboratory accident. It is of decisive importance to make the diagnosis as quickly as possible in order to limit any further spread of the disease.

This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the orderMononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication.

Introduction Precise recommendations on how to plan a training schedule for headers in soccer are rare. To ensure a clean technical and non-hazardous movement when heading the ball, specialized literature postulates the necessity of strengthening the muscles stabilizing the trunk. The question was how the muscles essential for headers would be working in non-fatigued and fatigued conditions. Therefore this study tried to record a potential temporal change of the maximum activity of m. sternocleidomastoideus (SCM) and m. trapezius pars descendens (TPD), after the muscles stabilizing the trunk (m. erector spinae and m. rectus abdominis) had been tired out by a treatment. Methods 12 nonprofessional soccer players were analyzed in a pre-post-test, performing straight headers using a pendulum header after a two footed take-off. The myoelectric potential of SCM and TPD was recorded with the help of telemetric surface electromyography. A foot pressure sensor was used to register the moment of take-off; an acceleration sensor was attached to the back of the head to register the ball contact. The treatment to tire out the trunk muscles included three abdominal excercises and two back excercises. After determining the moment of maximal activity within the defined time frame, that started at the point of take-off and ended at the point ob ball contact, pretest and posttest were compared with the help of a t-test to find out if any significant changes occured. Results The moments of maximum muscle activity of SCM and TPD changed heterogeneously but not significantly. Different muscular activation strategies could be identified. The maximum activity of TPD of four subjects occured in the posttest earlier (15% of the time frame). Three subjects showed an earlier activation of SCM (15% of the time frame), whereas five subjects didn9t show any differences at all. Specialized literature postulated a co-contraction of SCM and TPD at the moment of ball contact, but this result could not be found in our tests. In general SCM was inactive at that point in time. Conclusion Beside the small number of subjects in the survey the reason for the heterogeneous and non-significant activation might be the redundant functioning of the muscles. The different temporal activations of TPD and SCM can be understood as inter-individual motor patterns. The stronger drawing back movement and acceleration originating in the throat-neck-region can be seen as a compensation of the tired trunk muscles. The reason for the missing co-contraction of SCM at the moment of ball contact, could be the standardized test methods with the help of an immovable pendulum header.

In Germany soil remediation technologies are looking back to a development of 10 to 15 years. In the eighties ground examination standards and waste limits values have been defined which are forming the basis of all remediation methods. Today the classical cleaning technologies like soil washing and activated coal adsorption have just reached their physical limits. Additional technical proceedings have to improve cleaning efficiency and decomposition of waste.

In the present serologocal study 120 monkey sera from different species originating from the Philippines, China, Uganda and undetermined sources and several groups of human sera comprising a total of 1288 specimens from people living in Germany were examined for the presence of antibodies directed against filoviruses (Marburg virus, strain Musoke/Ebola virus, subtype Zaire, strain Mayinga/Reston virus). Sera were screened using a filovirus-specific enzyme-linked immunosorbent assay (ELISA). ELISA-positive sera were then confirmed by the indirect immunofluorescence technique, Western blot technique, and a blocking assay, and declared positive when at least one cornfirmation test was reactive. Altogether 43.3% of the monkey sera and 6.9% of the human sera reacted positively with at least one of the three different filovirus antigens. The blocking assays show that antibodies, detected in the sera, are directed to specific filovirus antigens and not caused by antigenic cross-reactivity with hitherto unknown agents. Data presented in this report suggest that subclinical filovirus infections may also occur in humans and in subhuman primates. They further suggest that filoviruses are not restricted to the African continent.

The low-frequency dielectric absorption of RbAg4l5 single crystals was found to have a maximum at about 1 kHz. It was tried to explain this by considering the occurrence of ordered regions of silver ions in the crystal structure, according to the domain model proposed by van Gool. Some aspects supporting this model are discussed.

The relative contribution of the vagus and splanchnic nerves as mediators of the action of 2-deoxy- D -glucose (2-DG) on the stomach and the pancreas is largely unknown. In consciou

The Marburg virus (MBGV) nucleocapsid complex is composed of four viral proteins (NP, L, VP35, and VP30) and the negative-strand nonsegmented genomic RNA. NP, L, and VP35 are functionally conserved among the order Mononegavirales, whereas VP30, a phosphoprotein, represents a filovirus-specific nucleocapsid protein. In the present paper, we have characterized the localization and function of VP30 phosphorylation. The main phosphorylation sites are represented by seven serine residues in the region of amino acid 40 to 51 of VP30. Additionally, trace amounts of phosphothreonine were detected. Substitution of serine residues 40 and 42 by alanine abolished the interaction of VP30 with NP-induced inclusion bodies, which contain nucleocapsid-like structures formed by NP. Substitution of the other phosphoserine residues had little effect on this interaction. Replacement of the introduced alanine residues 40 and 42 by aspartate restored the interaction between VP30 and the NP inclusions pointing to the importance of negative charges at these particular positions.

The nucleoprotein (NP) of Marburg virus is phosphorylated at serine and threonine residues in a ratio of 85:15, regardless of whether the protein is isolated from virions or from eukaryotic expression systems. Phosphotyrosine is absent. Although many potential phosphorylation sites are located in the N-terminal half of NP, this part of the protein is not phosphorylated. Analyses of phosphorylation state and phosphoamino acid content of truncated NPs expressed in HeLa cells using the vaccinia virus T7 expression system led to the identification of seven phosphorylated regions (region I*, amino acids 404–432; II*, amino acids 446–472; III*, amino acids 484–511; IV*, amino acids 534–543; V*, amino acid 549; VI*, amino acids 599–604; and VII*, amino acid 619) with a minimum of seven phosphorylated amino acid residues located in the C-terminal half of NP. All phosphothreonine residues and consensus recognition sequences for protein kinase CKII are located in regions I*–V*. Regions VI* and VII* contain only phosphoserine with three of four serine residues in consensus recognition motifs for proline-directed protein kinases. Mutagenesis of proline-adjacent serine residues to alanine or aspartic acid did not influence the function of NP in a reconstituted transcription/replication system; thus it is concluded that serine phosphorylation in the most C-terminal part of NP is not a regulatory factor in viral RNA synthesis.

Background. A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. Methods. The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcriptionpolymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. Results. The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged 74 years (90%) and low in patients aged 1019 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of

The 3′ and 5′ ends of Marburg virus (MBG)-specific mRNA species have been determined using reverse transcription-PCR, rapid amplification of cDNA ends, or the reverse ligation-mediated PCR procedure after removal of cap structures with tobacco acid pyrophosphatase. The polyadenylation sites of all MBG-specific mRNAs were strictly conserved and corresponded to the predicted transcriptional stop signals of genomic RNA. Determination of the 5′ ends of the mRNA species showed that mRNA synthesis started precisely at the first nucleotide of a highly conserved transcriptional start site. The 5′ ends of the mRNA species can build a stable secondary structure with the conserved nucleotides always located in the stem region of a hairpin. Nucleotide substitutions in the conserved 5′ regions are accompanied by compensatory mutations of the complementary nucleotide thus leading to a conservation of the secondary structures. Compensatory mutations were also found when 5′ ends of mRNA of MBG strain Musoke were compared with MBG strain Popp or the closely related Ebola virus, indicating that the secondary structures will be conserved even if the sequence is altered.

Balloon kyphoplasty (BKP) and vertebroplasty (VP) are clinically effective procedures. However, BKP has been occasionally associated with failure, although the cause has not been established thus far. We believe that, especially in patients with severe osteoporosis and osteonecrosis, BKP fails due to the so-called stress shielding effect and the stiffness of cement. In these patients, other bone-preserving kyphoplastic procedures and vertebroplasty, as well as recently introduced cements adjusted to the severity of osteoporosis, might be beneficial. Furthermore, it is essential to achieve complete straightening intraoperatively when performing BKP, because any persistent residual kyphosis will aggravate the burden on the adjacent vertebral bodies following the creation of a cavity filled with cement. Therefore, it would be meaningful to consider alternative bone-preserving kyphoplastic measures instead of BKP. In cases of older fractures, one should consider the use of VP and the recently introduced cements.

Upper Carboniferous sandstones in NW-Germany consist of thick successions of cyclic deposits and are major tight gas reservoirs. This study presents the heterogeneity exposed in a large quarry near Osnabrück, Germany, which contains faulted and jointed fourth-order coarse- to fine-grained tight sandstone cycles separated by anthracite coal seams. First, we characterize the rocks and the lateral variation of rock properties such as porosity, diagenesis and structural inventory. Than we test whether the quarry may act as a tight gas reservoir analogue to better constrain input data for reservoir modeling. The tight sandstones are intensely compacted and cemented with quartz and generally characterized by low matrix porosities < 8 % (He-pycnometry on plugs and cuttings) and very low permeabilities (< 0.01 mD). Porosity is generally secondary intraparticle, formed by detrital and authigenic carbonate dissolution and dissolution of feldspars. Matrix porosity significantly increases up to 25% in corridors around faults. Main rock types can be distinguished by spectral gamma ray in the quarry. Quartz veins and fault mineralizations indicate fluid flow within and around faults. Normal faults show bands of clay smear and gouge, forming compartments. Fractures were analyzed in a 50 x 50 m section of the quarry wall using LIDAR laser scanning. This digitized quarry face also allows the characterization of the lithology and quantitative measurement of bedding, fracture and fault orientation data in inaccessible areas. Our high-resolution field analogue enables a better understanding of tight sand reservoir properties and reservoir quality at a sub-seismic scale, considering both the change of porosity during diagenesis and the formation of structures. Results may be used to develop data-driven exploration strategies and improved development options for similar subsurface tight gas reservoirs, especially in Northern Germany.

In response to a recent outbreak in China, detection assays for a novel avian influenza A(H7N9) virus need to be implemented in a large number of public health laboratories. Here we present real-time reverse-transcription polymerase chain reaction (RT-PCR) assays for specific detection of this virus, along with clinical validation data and biologically-safe positive controls.