106 results on '"Gelderblom HR"'
Search Results
2. Helmut Ruska and the visualisation of viruses
- Author
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Kruger, DH, primary, Schneck, P, additional, and Gelderblom, HR, additional
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- 2000
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Catalog
3. Olfactory transmucosal SARS-CoV-2 invasion as a port of central nervous system entry in individuals with COVID-19.
- Author
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Meinhardt J, Radke J, Dittmayer C, Franz J, Thomas C, Mothes R, Laue M, Schneider J, Brünink S, Greuel S, Lehmann M, Hassan O, Aschman T, Schumann E, Chua RL, Conrad C, Eils R, Stenzel W, Windgassen M, Rößler L, Goebel HH, Gelderblom HR, Martin H, Nitsche A, Schulz-Schaeffer WJ, Hakroush S, Winkler MS, Tampe B, Scheibe F, Körtvélyessy P, Reinhold D, Siegmund B, Kühl AA, Elezkurtaj S, Horst D, Oesterhelweg L, Tsokos M, Ingold-Heppner B, Stadelmann C, Drosten C, Corman VM, Radbruch H, and Heppner FL more...
- Subjects
- Central Nervous System, Humans, RNA, Viral genetics, Smell physiology, Virus Internalization, Brain virology, COVID-19 virology, Olfactory Mucosa virology, SARS-CoV-2 pathogenicity
- Abstract
The newly identified severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a pandemic respiratory disease. Moreover, thromboembolic events throughout the body, including in the CNS, have been described. Given the neurological symptoms observed in a large majority of individuals with COVID-19, SARS-CoV-2 penetrance of the CNS is likely. By various means, we demonstrate the presence of SARS-CoV-2 RNA and protein in anatomically distinct regions of the nasopharynx and brain. Furthermore, we describe the morphological changes associated with infection such as thromboembolic ischemic infarction of the CNS and present evidence of SARS-CoV-2 neurotropism. SARS-CoV-2 can enter the nervous system by crossing the neural-mucosal interface in olfactory mucosa, exploiting the close vicinity of olfactory mucosal, endothelial and nervous tissue, including delicate olfactory and sensory nerve endings. Subsequently, SARS-CoV-2 appears to follow neuroanatomical structures, penetrating defined neuroanatomical areas including the primary respiratory and cardiovascular control center in the medulla oblongata. more...
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- 2021
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4. Rapid Viral Diagnosis of Orthopoxviruses by Electron Microscopy: Optional or a Must?
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Gelderblom HR and Madeley D
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- Animals, Exanthema diagnosis, Exanthema virology, Herpesviridae classification, Herpesviridae ultrastructure, Herpesviridae Infections diagnosis, Herpesviridae Infections virology, Humans, Orthopoxvirus classification, Poxviridae Infections prevention & control, Smallpox diagnosis, Smallpox virology, Microscopy, Electron methods, Orthopoxvirus ultrastructure, Poxviridae Infections diagnosis, Poxviridae Infections virology
- Abstract
Diagnostic electron microscopy (DEM) was an essential component of viral diagnosis until the development of highly sensitive nucleic acid amplification techniques (NAT). The simple negative staining technique of DEM was applied widely to smallpox diagnosis until the world-wide eradication of the human-specific pathogen in 1980. Since then, the threat of smallpox re-emerging through laboratory escape, molecular manipulation, synthetic biology or bioterrorism has not totally disappeared and would be a major problem in an unvaccinated population. Other animal poxviruses may also emerge as human pathogens. With its rapid results (only a few minutes after arrival of the specimen), no requirement for specific reagents and its "open view", DEM remains an important component of virus diagnosis, particularly because it can easily and reliably distinguish smallpox virus or any other member of the orthopoxvirus (OPV) genus from parapoxviruses (PPV) and the far more common and less serious herpesviruses (herpes simplex and varicella zoster). Preparation, enrichment, examination, internal standards and suitable organisations are discussed to make clear its continuing value as a diagnostic technique., Competing Interests: The authors declare no conflict of interests. The conclusions drawn are those of the authors and do not necessarily represent the views of our institutions. more...
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- 2018
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5. Ebola virus disease: any risk for oral and maxillo-facial surgery? An overview.
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Reichart PA, Gelderblom HR, Khongkhunthian P, and Schmidt-Westhausen A
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- Africa South of the Sahara, Africa, Western, Hemorrhagic Fever, Ebola epidemiology, Hemorrhagic Fever, Ebola virology, Humans, Medical Missions, Risk Factors, Disease Outbreaks, Hemorrhagic Fever, Ebola diagnosis, Hemorrhagic Fever, Ebola transmission, Infectious Disease Transmission, Patient-to-Professional, Surgery, Oral, Surgery, Plastic
- Abstract
The 2014-2015 outbreak of the Ebola virus disease (EVD) in West Africa has been considered a major global health emergency by the WHO. Implications for health care providers including oral and maxillo-facial surgeons have been published by the WHO, the Centers for Disease Control and Prevention (USA), and other medical societies and public health organizations. While the risk of infection with the Ebola virus seems to be rather small in Europe, maxillo-facial and plastic surgeons often travel to Africa to treat patients with facial burns, cleft-lip and palate, and noma. The likelihood of an encounter with patients infected by Ebola virus in subsaharan and West Africa, therefore, has increased during the last 2 years. The purpose of this short overview was to summarize the virology of the Ebola virus, transmission, epidemiology, clinical features, oral manifestations, treatment, and possible implications for maxillo-facial surgeons of EDV. more...
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- 2016
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6. [Lessons learnt from the German smallpox outbreaks after World War II].
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Sasse J and Gelderblom HR
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- Bioterrorism statistics & numerical data, Germany epidemiology, Humans, Incidence, Risk Factors, Smallpox diagnosis, Survival Rate, Bioterrorism prevention & control, Disease Outbreaks prevention & control, Disease Outbreaks statistics & numerical data, Smallpox mortality, Smallpox prevention & control, Smallpox Vaccine therapeutic use
- Abstract
Background: Even though smallpox was declared eradicated by WHO in 1980, it cannot be ruled out that the etiological variola virus could be used as a biological weapon. Undestroyed viruses from biowarfare programmes, virus strains left undetected in a freezer or dangerous recombinant poxvirus constructs could cause dangerous outbreaks in a relatively unprotected population., Objectives: Despite an abundance of studies performed during the eradication of smallpox, epidemiological data for preparedness planning and outbreak control in modern, industrialized countries are scarce., Material and Methods: Full-text hand search for the period from 1945 to 1975 in the main German public health journals., Results: After World War II 12 smallpox outbreaks occurred in Germany. They were studied with the focus on the period of contagiousness, the protective effect of vaccination, booster-effect of revaccination and the place of infection. A total of 95 individuals contracted smallpox, including 10 fatalities. Despite having been previously vaccinated, 81 vaccinated persons came down with smallpox, yet 91% of them developed only mild symptoms. These patients presented a high risk for spreading the infection to contact persons due to misinterpretation of symptoms and the continuing social contacts. Basically, the risk of transmission in the first 2 to 3 days after onset of symptoms was low, thus facilitating antiepidemic measures. The importance of hospital preparedness is emphasized by the fact that most infections occurred in hospitals., Conclusion: The data analyzed provide valuable information for today's outbreak response planning and counter bioterrorism preparedness. more...
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- 2015
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7. Electron microscopy in rapid viral diagnosis: an update.
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Gentile M and Gelderblom HR
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- Animals, History, 17th Century, History, 18th Century, History, 19th Century, History, 20th Century, Humans, Microscopy, Electron history, Virus Diseases virology, Viruses isolation & purification, Microscopy, Electron methods, Virus Diseases diagnosis, Viruses ultrastructure
- Abstract
Diagnostic electron microscopy (DEM) has conceptual predecessors â€" the application of the sense of vision and of light microscopy in medicine. The evolvement of DEM and the role of its two branches - histopathology and rapid negative-contrast DEM - are described in this review, with a focus on the latter. By its resolving power of 2 nm in praxi, DEM is able to visualize all kinds of pathogens, bacteria, parasites, even the smallest viruses. In contrast to other laboratory diagnostic methods, DEM excels by speed and "open view". All structures on the support grid can be assigned directly by "pattern recognition" of their fine structure to a specific family of agents. The morphology-based "catch-all" diagnosis can be decisive as a differential diagnosis and will help as a preliminary diagnosis to select and apply proper diagnostic tools for typing of the observed agent. Based on two case reports, the advantages and possible pitfalls of DEM are exemplified and hints are given to make DEM reliable and effective. Finally the role of DEM in medicine and the wider fields of life sciences are described together with the organizational conditions to guarantee its future in laboratory diagnostics. more...
- Published
- 2014
8. A novel fish herpesvirus of Osmerus eperlanus.
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Jakob NJ, Kehm R, and Gelderblom HR
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- Animals, Genome, Viral, Herpesviridae genetics, Herpesviridae Infections virology, Microscopy, Electron, Virion ultrastructure, Fish Diseases virology, Herpesviridae ultrastructure, Herpesviridae Infections veterinary, Osmeriformes virology
- Abstract
A herpesvirus of smelt (Osmerus eperlanus) was identified by thin section electron microscopy. Degenerated cells of skin lesions located on the back fin of smelt showed either intranucleic- or cytoplasmic herpesvirus-specific structures. In the nuclei "naked" virus capsids with a diameter of about 100 nm were observed. The diameter of the complete virion including its unilaterally extended envelope ranged from 200 to 350 nm. Remarkably, in complete virions the electron-opaque tegument is completely filling the region between nucleocapsid and envelope and as another unique feature the virion shows a "comet-shape" due to a long unilateral extension of its envelope. This kind of shape had been not reported for any of herpesviruses known so far. Consequently this virus was termed herpesvirus of Osmerus eperlanus (HVOE1) or Comet herpesvirus of smelt. Due to the long time storage at the nonstandard temperature of smelt virus the biological and genomic analysis of the HVOE1 was hampered. All attempts to study host range of HVOE1 failed as no virus replication was observed, indicating that infectivity was lost or the suitable cell culture was missing. The genomic DNA of HVOE1 was analyzed by DNA restriction endonucleases. more...
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- 2010
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9. WITHDRAWN: In Memoriam Toshiyuki Goto (1948-2007).
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Ogura H and Gelderblom HR
- Abstract
The publisher regrets that this article is an accidental duplication of an article that has already been published in JMIC, 38/7, pages 699 doi:10.1016/j.micron.2007.05.007. The duplicate article has therefore been withdrawn. more...
- Published
- 2007
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10. Arguments pro disinfection in diagnostic electron microscopy: a response to Madeley and Biel.
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Gelderblom HR, Bannert N, and Pauli G
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- Humans, Communicable Disease Control methods, Disease Transmission, Infectious prevention & control, Disinfection methods, Microscopy, Electron methods, Specimen Handling methods
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- 2007
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11. Reply to: disinfection in diagnostic electron microscopy prior to preparation?
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Gelderblom HR, Bannert N, and Pauli G
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- Humans, Communicable Disease Control methods, Disease Transmission, Infectious prevention & control, Disinfection methods, Microscopy, Electron methods, Specimen Handling methods
- Published
- 2007
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12. Pitfalls in diagnosing human poxvirus infections.
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Nitsche A, Gelderblom HR, Eisendle K, Romani N, and Pauli G
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- Adult, Agricultural Workers' Diseases diagnosis, Animals, Animals, Domestic, Humans, Male, Microscopy, Electron methods, Parapoxvirus, Poxviridae Infections virology, Agricultural Workers' Diseases virology, Occupational Exposure, Poxviridae Infections diagnosis, Zoonoses virology
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- 2007
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13. Morphological findings and energy dispersive X-ray microanalysis of oral amalgam tattoos.
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Zhang X, Gelderblom HR, Zierold K, and Reichart PA
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- Dental Amalgam chemistry, Dental Amalgam metabolism, Electron Probe Microanalysis, Foreign Bodies metabolism, Foreign Bodies pathology, Humans, Metals, Heavy adverse effects, Metals, Heavy analysis, Metals, Heavy metabolism, Microscopy, Electron, Mouth Mucosa metabolism, Mouth Mucosa pathology, Particle Size, Tattooing, Dental Amalgam adverse effects
- Abstract
Oral amalgam tattoos (AT) are distinct pigmentations of the oral mucosa resulting from accidental incorporation of dental amalgam in the oral soft tissues. Dental amalgams and in particular mercury, one of the constituents of dental amalgams, have for long been considered toxic. Oral ATs are easily accessible to study soft tissue reaction to amalgam and its degradation products. In this study, 17 oral ATs were examined by transmission electron microscopy and energy dispersive X-ray microanalysis. Ultrastructurally, in the ATs, three kinds of electron-dense particles were observed. The largest particles ranged in size from 0.5 up to several 100 microm. Smaller electron-dense inclusions (0.5-0.1 microm) were seen extracellularly associated with meshworks of elastic fibers and collagen bundles. The third and smallest type of particles (5-30 nm in diameter) was found with basement membranes of small vessels and pericytes and particularly decorating collagen bundles. Element analysis regularly revealed the presence of silver, sulphur, copper and lead in the AT decay products. Mercury was found in only one instance. Tissue reactions due to ATs seem to be minimal. No acute inflammatory changes were seen. Larger inclusions occasionally were surrounded by macrophages and multinucleated cells. TEM and element analysis may in specific cases be helpful in the differential diagnosis of pigmented lesions of the oral mucosa. more...
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- 2007
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14. Routine, rapid, noninvasive diagnosis of viral skin exanthems.
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Gelderblom HR, Bannert N, Muss W, and Madeley CR
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- Diagnosis, Differential, Humans, Microscopy, Electron, Viruses ultrastructure, Skin Diseases, Viral diagnosis
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- 2006
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15. Microwave-assisted tissue processing for same-day EM-diagnosis of potential bioterrorism and clinical samples.
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Schroeder JA, Gelderblom HR, Hauroeder B, Schmetz C, Milios J, and Hofstaedter F
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- Animals, Biopsy, Humans, Liver ultrastructure, Microwaves, Parasitic Diseases diagnosis, Time Factors, Virus Diseases diagnosis, Bioterrorism, Histological Techniques methods, Microscopy, Electron methods
- Abstract
The purpose of this study was to explore the turnaround times, section and image quality of a number of more "difficult" specimens destined for rapid diagnostic electron microscopy (EM) after microwave-assisted processing. The results were assessed and compared with those of conventionally processed samples. A variety of infectious agents, some with a potential for bioterrorism, and liver biopsies serving as an example for routine histopathology samples were studied. The samples represented virus-producing cell cultures (such as SARS-coronavirus, West Nile virus, Orthopox virus), bacteria suspensions (cultures of Escherichia coli and genetically knockout apathogenic Bacillus anthracis), suspensions of parasites (malaria Plasmodium falciparum, Leishmania major, Microsporidia cuniculi, Caenorhabditis elegans), and whole Drosophila melanogaster flies infected with microsporidia. Fresh liver samples and infected flies were fixed in Karnovsky-fixative by microwaving (20 min), all other samples were fixed in buffered glutaraldehyde or Karnovsky-fixative overnight or longer. Subsequently, all samples were divided to evaluate alternative processing protocols: one part of the sample was OsO4-postfixed, ethanol-dehydrated, Epon-infiltrated (overnight) in an automated tissue processor (LYNX, Leica), and polymerized at 60 degrees C for 48 h; in parallel the other part was microwave-assisted processed in the bench microwave device (REM, Milestone), including post-osmication and the resin block polymerization. The microwave-assisted processing protocol required at minimum 3 h 20 min: the respective epon resin blocks were uniformly polymerized allowing an easy sectioning of semi- and ultrathin sections. Sections collected on non-coated 200 mesh grids were stable in the electron beam and showed an excellent preservation of the ultrastructure and high contrast, thus allowing an easy, unequivocal and rapid assessment of specimens. Compared with conventional routine methods, microwave technology facilitates a significant reduction in sample processing time from days to hours without any loss in ultrastructural details. Microwave-assisted processing could, therefore, be a substantial benefit for the routine electron microscopic diagnostic workload. Due to its speed and robust performance it could be applied wherever a rapid electron microscopy diagnosis is required, e.g., if bioterrorism or emerging agents are suspected. Combining microwave technology with digital image acquisition, the 1-day diagnosis based on ultrathin section electron microscopy will become possible, with crucial or interesting findings being consulted or shared worldwide with experts using modern telemicroscopy tools via Internet. more...
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- 2006
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16. Molecular and biological characterization of human monoclonal antibodies binding to the spike and nucleocapsid proteins of severe acute respiratory syndrome coronavirus.
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van den Brink EN, Ter Meulen J, Cox F, Jongeneelen MA, Thijsse A, Throsby M, Marissen WE, Rood PM, Bakker AB, Gelderblom HR, Martina BE, Osterhaus AD, Preiser W, Doerr HW, de Kruif J, and Goudsmit J
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal genetics, Antibodies, Viral chemistry, Antibodies, Viral genetics, Antibodies, Viral immunology, Antibody Specificity, Binding Sites, Chlorocebus aethiops, Coronavirus Nucleocapsid Proteins, Epitope Mapping, Humans, Molecular Sequence Data, Neutralization Tests, Spike Glycoprotein, Coronavirus, Vero Cells, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal immunology, Membrane Glycoproteins immunology, Nucleocapsid Proteins immunology, Severe acute respiratory syndrome-related coronavirus immunology, Viral Envelope Proteins immunology
- Abstract
Human monoclonal antibodies (MAbs) were selected from semisynthetic antibody phage display libraries by using whole irradiated severe acute respiratory syndrome (SARS) coronavirus (CoV) virions as target. We identified eight human MAbs binding to virus and infected cells, six of which could be mapped to two SARS-CoV structural proteins: the nucleocapsid (N) and spike (S) proteins. Two MAbs reacted with N protein. One of the N protein MAbs recognized a linear epitope conserved between all published human and animal SARS-CoV isolates, and the other bound to a nonlinear N epitope. These two N MAbs did not compete for binding to SARS-CoV. Four MAbs reacted with the S glycoprotein, and three of these MAbs neutralized SARS-CoV in vitro. All three neutralizing anti-S MAbs bound a recombinant S1 fragment comprising residues 318 to 510, a region previously identified as the SARS-CoV S receptor binding domain; the nonneutralizing MAb did not. Two strongly neutralizing anti-S1 MAbs blocked the binding of a recombinant S fragment (residues 1 to 565) to SARS-CoV-susceptible Vero cells completely, whereas a poorly neutralizing S1 MAb blocked binding only partially. The MAb ability to block S1-receptor binding and the level of neutralization of the two strongly neutralizing S1 MAbs correlated with the binding affinity to the S1 domain. Finally, epitope mapping, using recombinant S fragments (residues 318 to 510) containing naturally occurring mutations, revealed the importance of residue N479 for the binding of the most potent neutralizing MAb, CR3014. The complete set of SARS-CoV MAbs described here may be useful for diagnosis, chemoprophylaxis, and therapy of SARS-CoV infection and disease. more...
- Published
- 2005
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17. Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48.
- Author
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Beutin L, Strauch E, Zimmermann S, Kaulfuss S, Schaudinn C, Männel A, and Gelderblom HR
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- Amino Acid Sequence, Antigens, Bacterial chemistry, Cloning, Molecular, Escherichia coli classification, Escherichia coli cytology, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Flagellin chemistry, Gene Expression Regulation, Bacterial, Molecular Sequence Data, Phylogeny, Sequence Alignment, Sequence Homology, Amino Acid, Serotyping, Antigens, Bacterial genetics, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins metabolism, Flagellin genetics, Flagellin metabolism, Genes, Bacterial genetics
- Abstract
Background: Serotyping of O-(lipopolysaccharide) and H-(flagellar) antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies., Results: The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) was determined and both were found 99.3% (1043 of 1050 nucleotides) identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella., Conclusion: The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strains which were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of different molecular weigh are functionally expressed and coassembled in the same flagellar filament in E. coli. more...
- Published
- 2005
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18. Rapid viral diagnosis: role of electron microscopy.
- Author
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Gentile M and Gelderblom HR
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- Forecasting, Humans, Specimen Handling, Staining and Labeling, Viruses isolation & purification, Microscopy, Electron, Virus Diseases diagnosis, Viruses ultrastructure
- Abstract
Starting in the 1960, electron microscopy (EM) became widely applied also in viral diagnosis. During the 1970th and 80th, many new agents were characterized from diagnostic cell cultures and clinical specimens. The wide introduction of ELISA- and PCR-techniques as well as cost-arguments recently reduced the role of EM in routine viral diagnosis. Compared to other diagnostic techniques, however, EM excells by speed and "open view", i.e. by the ability to detect also the "un-expected" without the need for specific reagents. As shown in 2003 by the elucidation of the SARS pandemia and the human monkeypox outbreak in US, EM is well suited as a safe, front-line diagnostic method in infectious diseases emergencies and/or in possible bioterrorist attacks. more...
- Published
- 2005
19. Functional comparison of the two gene products of Thogoto virus segment 6.
- Author
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Hagmaier K, Gelderblom HR, and Kochs G
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- Animals, Cricetinae, Humans, Interferon-beta biosynthesis, Mice, Replicon, Thogotovirus genetics, Virion physiology, Virus Replication, Interferon-beta antagonists & inhibitors, Thogotovirus physiology, Viral Matrix Proteins physiology, Viral Structural Proteins physiology
- Abstract
The sixth genomic segment of Thogoto virus (THOV) encodes two proteins, the viral matrix protein (M) and an accessory protein with an interferon (IFN)-antagonistic function named ML. M and ML are shown in this study to be structural components of the virion. Using an in vivo system based on the reconstitution of functional THOV ribonucleoprotein complexes from cloned cDNAs, it was demonstrated that M has an inhibitory effect on the viral RNA-dependent RNA polymerase (RdRP) and is essential for the formation of virus-like particles (VLPs). The functional domain responsible for the regulation of RdRP activity resides within the C-terminal half of M, while full-length M protein is required for VLP formation. The ML protein cannot complement M with respect to either RdRP downregulation or particle formation, although it is identical to M apart from a 38 aa extension at the C terminus. In contrast, ML, but not M, is able to prevent the induction of IFN-beta by double-stranded RNA. This function is contained within the C-terminal half of ML. These data suggest major structural differences between M and ML that could explain the different activities of the two proteins. more...
- Published
- 2004
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20. Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets.
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ter Meulen J, Bakker AB, van den Brink EN, Weverling GJ, Martina BE, Haagmans BL, Kuiken T, de Kruif J, Preiser W, Spaan W, Gelderblom HR, Goudsmit J, and Osterhaus AD
- Subjects
- Animals, Female, Ferrets, Humans, Immunization, Passive, Immunoglobulin G immunology, Lung pathology, Lung virology, Severe acute respiratory syndrome-related coronavirus immunology, Severe acute respiratory syndrome-related coronavirus isolation & purification, Severe Acute Respiratory Syndrome pathology, Severe Acute Respiratory Syndrome virology, Virus Replication, Antibodies, Monoclonal therapeutic use, Severe Acute Respiratory Syndrome prevention & control
- Abstract
SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal antibody in ferrets, which can be readily infected with the virus. Prophylactic administration of the monoclonal antibody at 10 mg/kg reduced replication of SARS coronavirus in the lungs of infected ferrets by 3.3 logs (95% CI 2.6-4.0 logs; p<0.001), completely prevented the development of SARS coronavirus-induced macroscopic lung pathology (p=0.013), and abolished shedding of virus in pharyngeal secretions. The data generated in this animal model show that administration of a human monoclonal antibody might offer a feasible and effective prophylaxis for the control of human SARS coronavirus infection. more...
- Published
- 2004
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21. An amino-terminal segment of hantavirus nucleocapsid protein presented on hepatitis B virus core particles induces a strong and highly cross-reactive antibody response in mice.
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Geldmacher A, Skrastina D, Petrovskis I, Borisova G, Berriman JA, Roseman AM, Crowther RA, Fischer J, Musema S, Gelderblom HR, Lundkvist A, Renhofa R, Ose V, Krüger DH, Pumpens P, and Ulrich R
- Subjects
- Animals, Antibodies, Viral immunology, Cross Reactions, Cryoelectron Microscopy, Female, Orthohantavirus classification, Hantavirus Infections prevention & control, Hepatitis B Core Antigens chemistry, Hepatitis B Core Antigens genetics, Mice, Mice, Inbred BALB C, Mice, Inbred C57BL, Nucleocapsid Proteins chemistry, Nucleocapsid Proteins genetics, Recombinant Fusion Proteins genetics, Viral Vaccines administration & dosage, Antibodies, Viral blood, Orthohantavirus immunology, Hepatitis B Core Antigens immunology, Nucleocapsid Proteins immunology, Recombinant Fusion Proteins immunology, Viral Vaccines immunology
- Abstract
Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90% T = 3 and 10% T = 4 shells. A map computed from T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains. more...
- Published
- 2004
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22. First international quality assurance study on the rapid detection of viral agents of bioterrorism.
- Author
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Niedrig M, Schmitz H, Becker S, Günther S, ter Meulen J, Meyer H, Ellerbrok H, Nitsche A, Gelderblom HR, and Drosten C
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- DNA, Viral blood, Ebolavirus genetics, Europe, Humans, Laboratories, Lassa virus genetics, Marburgvirus genetics, Orthopoxvirus genetics, Quality Control, Virus Diseases diagnosis, Virus Diseases virology, Bioterrorism, Ebolavirus isolation & purification, International Cooperation, Lassa virus isolation & purification, Marburgvirus isolation & purification, Orthopoxvirus isolation & purification, Polymerase Chain Reaction methods
- Abstract
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 > or = 5,000 and > or = 100,000 copies per ml, respectively. Sensitivity levels were not significantly different between viruses. False-negative results were attributable to a lack of sensitivity. more...
- Published
- 2004
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23. Detection of human polyomaviruses in urine from bone marrow transplant patients: comparison of electron microscopy with PCR.
- Author
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Biel SS, Nitsche A, Kurth A, Siegert W, Ozel M, and Gelderblom HR
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- BK Virus genetics, Coloring Agents, Humans, JC Virus genetics, JC Virus isolation & purification, Metals, Heavy, Microscopy, Electron, Polymerase Chain Reaction, Polyomavirus genetics, Polyomavirus ultrastructure, Sensitivity and Specificity, Ultracentrifugation, Urine virology, Bone Marrow Transplantation, Polyomavirus isolation & purification
- Abstract
Background: We studied electron microscopy (EM) as an appropriate test system for the detection of polyomavirus in urine samples from bone marrow transplant patients., Methods: We evaluated direct EM, ultracentrifugation (UC) before EM, and solid-phase immuno-EM (SPIEM). The diagnostic accuracy of EM was studied by comparison with a real-time PCR assay on 531 clinical samples., Results: The detection rate of EM was increased by UC and SPIEM. On 531 clinical urine samples, the diagnostic sensitivity of EM was 47% (70 of 149) with a specificity of 100%. We observed a linear relationship between viral genome concentration and the proportion of urine samples positive by EM, with a 50% probability for a positive EM result for urine samples with a polyomavirus concentration of 10(6) genome-equivalents (GE)/mL; the probability of a positive EM result was 0% for urine samples with <10(3) GE/mL and 100% for urine samples containing 10(9) GE/mL., Conclusions: UC/EM is rapid and highly specific for polyomavirus in urine. Unlike real-time PCR, EM has low sensitivity and cannot quantify the viral load. more...
- Published
- 2004
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24. Type 1 diabetes in Swedish bank voles (Clethrionomys glareolus): signs of disease in both colonized and wild cyclic populations at peak density.
- Author
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Niklasson B, Hörnfeldt B, Nyholm E, Niedrig M, Donoso-Mantke O, Gelderblom HR, and Lernmark A
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- Animals, Arvicolinae, Diabetes Mellitus, Type 1 physiopathology, Population Dynamics, Diabetes Mellitus, Type 1 veterinary
- Abstract
Colonized bank voles (Clethrionomys glareolus) originating from Sweden developed type 1 diabetes. Animals became polydipsic, glucosuric, and hyperglycemic and gradually developed a lethal ketoacidosis. Pancreas in animals with end-stage disease showed total destruction of islet cells. Interestingly, also a high proportion of wild bank voles in cyclic populations that were trapped at (or close to) the cyclic population density peak frequently showed high blood glucose levels and pathological glucose tolerance test. Extensive islet destruction was not seen in wild bank voles at the time of capture, but did develop in some of the animals over a time period of two months. Diabetes in both colonized and wild bank voles was associated with Ljungan virus (LV). LV could be isolated from the pancreas of diabetic bank voles and antigen detected at the site of tissue damage by immunohistochemistry. In addition, picornavirus-like particles were visualized in the islets of diabetic voles using thin-section transmission electron microscopy. more...
- Published
- 2003
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25. Isolation of Vibrio vulnificus and atypical Vibrio from surface water of the Baltic Sea in Germany.
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Jores J, Stephan R, Knabner D, Gelderblom HR, and Lewin A
- Subjects
- Animals, Base Sequence, DNA, Ribosomal chemistry, Germany, Phylogeny, RNA, Ribosomal, 16S genetics, Species Specificity, Vibrio classification, Vibrio genetics, Vibrio pathogenicity, Virulence, Eels microbiology, Fish Diseases microbiology, Vibrio isolation & purification, Vibrio vulnificus isolation & purification, Water Microbiology
- Abstract
From 1995 to 1997 several defined species like V. alginolyticus, V. anguillarum, V. cholerae (non O1 and non O139), V. mimicus, V. parahaemolyticus and V. vulnificus were isolated during a survey to determine the presence of V. vulnificus in the brackish water of the Baltic Sea in Germany. Moreover atypical Vibrio isolates were detected. Four isolates belonging to a group of atypical Vibrio and possibly representing a new species in the genus Vibrio were characterized in detail. All four strains were isolated from surface costal waters. Based on 16S rDNA sequence analysis they showed the highest relatedness to the species V. navarrensis and V. vulnificus. The strains did not harbor the species specific hemolysin gene vvhA from V. vulnificus as shown by PCR and hybridization experiments. Moreover, they differed in at least two biochemical parameters tested from the hitherto described Vibrio species. All these strains induced hemolysis on washed blood agar dishes and showed phase variations on Luria Bertani agar dishes. Because of the similarity to the eel pathogen V. vulnificus, we infected eels with one of the four atypical strains (CH-291), but no pathogenicity for eels could be detected. Furthermore, Vero cell tests with supernatants of bacterial cultures did not reveal secreted Vero cell cytotoxic compounds. This indicates a nonpathogenic nature of these strains. more...
- Published
- 2003
26. Replication-competent hybrids between murine leukemia virus and foamy virus.
- Author
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Shikova-Lekova E, Lindemann D, Pietschmann T, Juretzek T, Rudolph W, Herchenröder O, Gelderblom HR, and Rethwilm A
- Subjects
- Amino Acid Sequence, Animals, DNA, Viral, Humans, Hybridization, Genetic, Leukemia Virus, Murine physiology, Leukemia Virus, Murine ultrastructure, Molecular Sequence Data, Mutation, Spumavirus physiology, Spumavirus ultrastructure, Leukemia Virus, Murine genetics, Spumavirus genetics, Virus Replication genetics
- Abstract
Replication-competent chimeric retroviruses constructed of members of the two subfamilies of Retroviridae, orthoretroviruses and spumaretroviruses, specifically murine leukemia viruses (MuLV) bearing hybrid MuLV-foamy virus (FV) envelope (env) genes, were characterized. All viruses had the cytoplasmic tail of the MuLV transmembrane protein. In ESL-1, a truncated MuLV leader peptide (LP) was fused to the complete extracellular portion of FV Env, and ESL-2 to -4 contained the complete MuLV-LP followed by N-terminally truncated FV Env decreasing in size. ESL-1 to -4 had an extended host cell range compared to MuLV, induced a cytopathology reminiscent of FVs, and exhibited an ultrastructure that combined the features of the condensed core of MuLV with the prominent surface knobs of FVs. Replication of ESL-2 to -4 resulted in the acquisition of a stop codon at the N terminus of the chimeric Env proteins. This mutation rendered the MuLV-LP nonfunctional and indicated that the truncated FV-LP was sufficient to direct Env synthesis into the secretory pathway. Compared to the parental viruses, the chimeras replicated with only moderate cell-free titers. more...
- Published
- 2003
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27. Electron microscopy for rapid diagnosis of infectious agents in emergent situations.
- Author
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Hazelton PR and Gelderblom HR
- Subjects
- Humans, Communicable Diseases, Emerging diagnosis, Microscopy, Electron, Specimen Handling methods, Virus Diseases diagnosis
- Abstract
Diagnostic electron microscopy has two advantages over enzyme-linked immunosorbent assay and nucleic acid amplification tests. After a simple and fast negative stain preparation, the undirected, "open view" of electron microscopy allows rapid morphologic identification and differential diagnosis of different agents contained in the specimen. Details for efficient sample collection, preparation, and particle enrichment are given. Applications of diagnostic electron microscopy in clinically or epidemiologically critical situations as well as in bioterrorist events are discussed. Electron microscopy can be applied to many body samples and can also hasten routine cell culture diagnosis. To exploit the potential of diagnostic electron microscopy fully, it should be quality controlled, applied as a frontline method, and be coordinated and run in parallel with other diagnostic techniques. more...
- Published
- 2003
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28. Avian paramyxovirus serotype 1 isolates from the spinal cord of parrots display a very low virulence.
- Author
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Grund CH, Werner O, Gelderblom HR, Grimm F, and Kösters J
- Subjects
- Animals, Antibodies, Monoclonal immunology, Chickens virology, Female, Germany epidemiology, Hemagglutination Tests veterinary, Male, Newcastle disease virus isolation & purification, Newcastle disease virus ultrastructure, Ovum virology, Serotyping, Specific Pathogen-Free Organisms, Spinal Cord virology, Virulence Factors, Newcastle Disease epidemiology, Newcastle Disease virology, Newcastle disease virus immunology, Newcastle disease virus pathogenicity, Psittaciformes virology
- Abstract
The spinal cord of 32 psittacines suffering from proventricular dilatation disease (PDD) was investigated. In six cases, a virus was isolated which upon electron microscopic examination revealed morphological details typical of members of the Paramyxoviridae. All isolates were subsequently characterized as avian paramyxovirus serotype 1 (APMV-1) by type-specific polyclonal antisera. According to their reactivity with APMV-1 specific monoclonal antibodies, the six isolates shared epitopes within the haemagglutinin-neuraminidase spike protein, distinct from pigeon-type paramyxoviruses and the LaSota vaccine strain. This grouping was further corroborated by properties of the haemagglutinin: all isolates showed a very thermosensitive haemagglutination activity and were rapid eluters. Virulence of the APMV-1 isolates in 1-day-old specific pathogen free (spf) chicken was very low, with intracerebral pathogenicity indices between 0 and 0.1. In embryonated spf chicken eggs, psittacine isolates replicated to high titres (10(8.6)-10(10.7) EID50/ml). However, they exhibited a reduced lethality over an observation time of 7 days (10(6.1)-10(8.3) ELD50/ml). In a haemagglutination inhibition test with parrot sera from birds with no history of APMV-1 vaccination, sera reacted preferentially with two isolates compared with APMV-1 vaccine strains LaSota and B1. The other four isolates exhibited a differentiated reaction pattern with the parrot sera, indicating an antigenic inhomogeneity. This is the first report of isolating very low virulent APMV-1 from neuronal tissue of parrots and implications for a possible role in slow progressing disease will be discussed. more...
- Published
- 2002
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29. Case Report. Oro-facial manifestations of Penicillium marneffei infection in a Thai patient with AIDS.
- Author
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Khongkunthian P, Isaratanan W, Samaranayake LP, Gelderblom HR, and Reichart PA
- Subjects
- Adult, Dermatomycoses drug therapy, Humans, Male, Mycoses complications, Thailand, AIDS-Related Opportunistic Infections microbiology, Dermatomycoses microbiology, Mycoses microbiology, Penicillium isolation & purification
- Abstract
Penicillium marneffei infection is prevalent in South-East Asia and Southern China and has been considered an acquired immunodeficiency syndrome (AIDS)-defining disease. This report focuses on the oral and facial manifestations of P. marneffei infection in a 28-year-old Thai male patient with AIDS. The clinical, mycological and ultrastructural features are described. more...
- Published
- 2002
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30. Comparative analysis of the genome and host range characteristics of two insect iridoviruses: Chilo iridescent virus and a cricket iridovirus isolate.
- Author
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Jakob NJ, Kleespies RG, Tidona CA, Müller K, Gelderblom HR, and Darai G
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Iridovirus isolation & purification, Iridovirus physiology, Microscopy, Electron, Molecular Sequence Data, Sequence Analysis, DNA, Viral Proteins genetics, Genome, Viral, Gryllidae virology, Iridovirus genetics, Iridovirus pathogenicity
- Abstract
The iridovirus isolate termed cricket iridovirus (CrIV) was isolated in 1996 from Gryllus campestris L. and Acheta domesticus L. (both Orthoptera, Gryllidae). CrIV DNA shows distinct DNA restriction patterns different from those known for Insect iridescent virus type 6 (IIV-6). This observation led to the assumption that CrIV might be a new species within the family Iridoviridae. CrIV can be transmitted perorally to orthopteran species, resulting in specific, fatal diseases. These species include Gryllus bimaculatus L. and the African migratory locust Locusta migratoria migratorioides (Orthoptera, Acrididae). Analysis of genomic and host range properties of this isolate was carried out in comparison to those known for IIV-6. Host range studies of CrIV and IIV-6 revealed no differences in the peroral susceptibility in all insect species and developmental stages tested to date. Different gene loci of the IIV-6 genome were analyzed, including the major capsid protein (274L), thymidylate synthase (225R), an exonuclease (012L), DNA polymerase (037L), ATPase (075L), DNA ligase (205R) and the open reading frame 339L, which is homologous to the immediate-early protein ICP-46 of frog virus 3. The average identity of the selected viral genes and their gene products was found to be 95.98 and 95.18% at the nucleotide and amino acid level, respectively. These data led to the conclusion that CrIV and IIV-6 are not different species within the Iridoviridae family and that CrIV must be considered to be a variant and/or a novel strain of IIV-6. more...
- Published
- 2002
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31. Stop codon insertion restores the particle formation ability of hepatitis B virus core-hantavirus nucleocapsid protein fusions.
- Author
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Kazaks A, Lachmann S, Koletzki D, Petrovskis I, Dislers A, Ose V, Skrastina D, Gelderblom HR, Lundkvist A, Meisel H, Borisova G, Krüger DH, Pumpens P, and Ulrich R
- Subjects
- Animals, Base Sequence, Hepatitis B Core Antigens immunology, Immunization, Mice, Mice, Inbred BALB C, Molecular Sequence Data, Nucleocapsid immunology, Nucleocapsid Proteins, Codon, Terminator, Hepatitis B Core Antigens genetics, Hepatitis B virus physiology, Nucleocapsid genetics, Recombinant Fusion Proteins immunology, Virion physiology
- Abstract
In recent years, epitopes of various origin have been inserted into the core protein of hepatitis B virus (HBc), allowing the formation of chimeric HBc particles. Although the C-terminus of a C-terminally truncated HBc (HBc) tolerates the insertion of extended foreign sequences, the insertion capacity is still a limiting factor for the construction of multivalent vaccines. Previously, we described a new system to generate HBc mosaic particles based on a read-through mechanism in an Escherichia coli suppressor strain [J Gen Virol 1997;78:2049-2053]. Those mosaic particles allowed the insertion of a 114-amino acid (aa)-long segment of a Puumala hantavirus (PUUV) nucleocapsid (N) protein. To study the value and the potential limitations of the mosaic approach in more detail, we investigated the assembly capacity of 'non-mosaic' HBc fusion proteins and the corresponding mosaic constructs carrying 94, 213 and 433 aa of the hantaviral N protein. Whereas the fusion proteins carrying 94, 114, 213 or 433 aa were not assembled into HBc particles, or only at a low yield, the insertion of a stop codon-bearing linker restored the ability to form particles with 94, 114 and 213 foreign aa. The mosaic particles formed exhibited PUUV-N protein antigenicity. Immunization of BALB/c mice with these mosaic particles carrying PUUV-N protein aa 1-114, aa 1-213 and aa 340-433, respectively, induced HBc-specific antibodies, whereas PUUV-N protein-specific antibodies were detected only in mice immunized with particles carrying N-terminal aa 1-114 or aa 1-213 of the N protein. Both the anti-HBc and anti-PUUV antibody responses were IgG1 dominated. In conclusion, stop codon suppression allows the formation of mosaic core particles carrying large-sized and 'problematic', e.g. hydrophobic, hantavirus sequences. more...
- Published
- 2002
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32. Brackiella oedipodis gen. nov., sp. nov., gram-negative, oxidase-positive rods that cause endocarditis of cotton-topped tamarin (Saguinus oedipus).
- Author
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Willems A, Gilhaus H, Beer W, Mietke H, Gelderblom HR, Burghardt B, Voigt W, and Reissbrodt R
- Subjects
- Animals, Bacterial Proteins analysis, Betaproteobacteria isolation & purification, DNA, Ribosomal analysis, Endocarditis, Bacterial microbiology, Endocarditis, Bacterial pathology, Fatty Acids analysis, Gram-Negative Bacterial Infections microbiology, Gram-Negative Bacterial Infections veterinary, Lipopolysaccharides analysis, Molecular Sequence Data, Myocardium pathology, Phenotype, RNA, Ribosomal, 16S genetics, Sequence Analysis, DNA, Siderophores metabolism, Spectroscopy, Fourier Transform Infrared, Betaproteobacteria classification, Betaproteobacteria enzymology, Endocarditis, Bacterial veterinary, Monkey Diseases microbiology, Oxidoreductases metabolism, Saguinus
- Abstract
A gram-negative, oxidase-positive, rod-shaped bacterium isolated from the heart of a cotton-topped tamarin was characterized by 16S rDNA sequence analysis, SDS-PAGE of whole-cell proteins, fatty acid analysis and biochemical tests. Outer-membrane proteins, iron-regulated outer-membrane proteins, lipopolysaccharides and siderophore production were studied. On the basis of the results, the organism belongs to the beta-Proteobacteria where it forms a separate line of descent, for which a novel genus and species are proposed, Brackiella oedipodis (LMG 19451T = DSM 13743T = NCIMB 13739T). Nearest phylogenetic neighbours of the new genus are Taylorella, Pelistega, Bordetella, Alcaligenes and Achromobacter. more...
- Published
- 2002
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33. Shammah-induced oral leukoplakia-like lesions.
- Author
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Zhang X, Schmitz W, Gelderblom HR, and Reichart PA
- Subjects
- Adult, Follow-Up Studies, Humans, Leukoplakia, Oral pathology, Male, Leukoplakia, Oral etiology, Plants, Toxic, Tobacco, Smokeless adverse effects
- Abstract
A Shammah-induced oral leukoplakia-like lesion is described in a 44-year-old Algerian patient, who used this specific chewing tobacco since 33 years. The extended white lesion was located to the right mandibular vestibule and had a homogeneous appearance. Shammah is a chewing tobacco consisting of powdered tobacco leaves with carbonate of lime and other substances. It has been associated with oral cancer in Saudi Arabia. Histologically, acanthosis, hyperortho- and parakeratosis were seen. The spinous cell layer showed large pale staining epithelial cells with pycnotic nuclei. Atypia was not observed, however, an increase in mitotic activity was apparent. The subepithelial infiltrate was mild. Electron microscopy showed changes in the basal membrane with interruptions, duplications and triplications. Follow-up of the patient for 2 years revealed that, whenever, the patient changed the location of application, the white lesion regressed or disappeared within 4-6 weeks. Due to the composition of Shammah, the lesion induced has features of a mucosal burn. In contrast to other smokeless tobacco variants, Shammah seems to cause changes which, according to the small number of reports, may transform into oral cancer. As such, Shammah-induced oral leukoplakia-like lesions may be considered precancerous. more...
- Published
- 2001
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34. Puumala (PUU) hantavirus strain differences and insertion positions in the hepatitis B virus core antigen influence B-cell immunogenicity and protective potential of core-derived particles.
- Author
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Koletzki D, Lundkvist A, Sjölander KB, Gelderblom HR, Niedrig M, Meisel H, Krüger DH, and Ulrich R
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Viral immunology, Arvicolinae, Epitopes, B-Lymphocyte immunology, Escherichia coli, Orthohantavirus classification, Hantavirus Infections immunology, Molecular Sequence Data, Nucleocapsid Proteins, Species Specificity, Vaccines, Synthetic chemistry, Vaccines, Synthetic genetics, Vaccines, Synthetic immunology, B-Lymphocytes immunology, Orthohantavirus immunology, Hantavirus Infections prevention & control, Hepatitis B Core Antigens immunology, Hepatitis B virus immunology, Nucleocapsid immunology
- Abstract
Hepatitis B virus (HBV) core-derived chimeric particles carrying a Puumala (PUU) hantavirus (strain Vranica/Hällnäs) nucleocapsid (N) protein sequence (aa 1-45), alternatively inserted at three distinct positions (N-, C-terminus, or the internal region), and mosaic particles consisting of HBV core as well as core/PUU (Vranica/Hällnäs) N (aa 1-45) readthrough protein were generated. Chimeric particles carrying the insert at the N-terminus or the internal region of core induced some protective immune response in bank voles (Clethrionomys glareolus) against a subsequent PUU virus (strain Kazan) challenge; 40-50% of the animals showed markers of protection. In contrast, internal insertion of PUU strain CG18-20 N (aa 1-45) into the HBV core caused a highly protective immune response in the bank vole model. Immunizations with particles carrying aa 75-119 of PUU (CG18-20) N at the C-terminus of core verified the presence of a second, minor protective region in the N protein. A strong PUU N-specific antibody response was detected not only in bank voles immunized with chimeric particles containing internal and N-terminal fusions of PUU N protein but also in animals immunized with the corresponding mosaic particles. Except for the exclusive occurrence of antibodies directed against aa 231-240 of N in non-protected animals post virus challenge, there was no additional obvious difference in the epitope-specificity of N-specific antibodies from immunized animals prior and post virus challenge., (Copyright 2000 Academic Press.) more...
- Published
- 2000
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35. Rapid quantification and differentiation of human polyomavirus DNA in undiluted urine from patients after bone marrow transplantation.
- Author
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Biel SS, Held TK, Landt O, Niedrig M, Gelderblom HR, Siegert W, and Nitsche A
- Subjects
- BK Virus isolation & purification, Calibration, DNA Primers, DNA, Viral urine, Humans, JC Virus isolation & purification, Plasmids, Reference Values, Reproducibility of Results, Time Factors, Transplantation, Homologous, BK Virus classification, Bone Marrow Transplantation, JC Virus classification, Polymerase Chain Reaction methods, Urine virology
- Abstract
A combined PCR assay was developed for the detection and typing of human polyomavirus (huPoV) in clinical samples, consisting of (i) a qualitative seminested PCR assay (snPCR) to discriminate between huPoV BK and JC and (ii) a high-throughput, quantitative TaqMan PCR assay (TM-PCR) for the general detection of huPoV. The TM-PCR detects huPoV DNA in a linear range from 10(7) to 10(1) copies per assay. In reproducibility runs, the inter- and intra-assay variabilities were < or =60 and < or =50%, respectively. The snPCR assay uses a set of four primers for the same region of the BK and JC viral genomes. In the first round of amplification, two general primers were used; in the second round, one of these general primers and two additional, BK- or JC-specific primers were used simultaneously to produce amplicons of different sizes specific for BK virus (246 bp) and JC virus (199 bp), respectively. We tested different urine dilutions in order to determine the inhibitory effects of urine on PCR amplification. Furthermore, we compared the use of native urine with DNA purified by different preparation procedures. Our results show, that a 1:10 dilution of the urine led to complete reduction of the amplification inhibition found with 6% of undiluted urine samples. In a clinical study including 600 urine specimens, our assay turned out to be fast, cheap, and reliable in both qualitative and quantitative aspects. more...
- Published
- 2000
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- View/download PDF
36. Specimen collection for electron microscopy.
- Author
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Gelderblom HR and Hazelton PR
- Subjects
- Humans, Microscopy, Electron methods, Specimen Handling methods, Virus Diseases diagnosis
- Published
- 2000
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37. Sendai virosomes revisited: reconstitution with exogenous lipids leads to potent vehicles for gene transfer.
- Author
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Ponimaskin E, Bareesel KK, Markgraf K, Reszka R, Lehmann K, Gelderblom HR, Gawaz M, and Schmidt MF
- Subjects
- Animals, Cells, Cultured, Chickens, DNA administration & dosage, HN Protein metabolism, Hemolysis, Microscopy, Electron, Gene Transfer Techniques, Genetic Vectors, Polylysine, Respirovirus, Viral Envelope Proteins genetics
- Abstract
A reliable new procedure is described for the reconstitution of Sendai viral envelopes suitable for gene transfer. Both fusion and hemagglutinin-neuraminidase glycoproteins were extracted from purified Sendai virus and reconstituted together with DNA in the presence of cholesterol:sphingomyelin:phosphatidylcholine:phosphatidylethanolamin e (Chol:SM:PC:PE) in a molar ratio of 3.5:3.5:2:1. Before reconstitution, the DNA to be transferred was condensed by pretreatment with polylysine. Exogenous lipid addition and the DNA-condensation step were essential for maximal size as well as for fusogenic activity of the resulting virosomes, the analysis of which revealed (1) the absence of any genomic material originating from Sendai virus, (2) the presence of fusogenic spikes in a functional orientation, (3) the encapsulation of reporter genes, and (4) high-transfer activity for plasmids carrying the green fluorescent protein (GFP) gene and double-stranded nucleotides into different mammalian cells. Transfer rates were up to 10-fold higher than those obtained with different cationic lipids. Gene delivery by means of our lipid-enriched Sendai virosomes extends the known gene transfer strategies, including those based on Sendai virus previously published., (Copyright 2000 Academic Press.) more...
- Published
- 2000
- Full Text
- View/download PDF
38. New chimaeric hepatitis B virus core particles carrying hantavirus (serotype Puumala) epitopes: immunogenicity and protection against virus challenge.
- Author
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Ulrich R, Koletzki D, Lachmann S, Lundkvist A, Zankl A, Kazaks A, Kurth A, Gelderblom HR, Borisova G, Meisel H, and Krüger DH
- Subjects
- Amino Acid Sequence, Animals, Antigens, Viral genetics, Arvicolinae, Base Sequence, Biotechnology, Epitopes genetics, Genetic Vectors, Orthohantavirus genetics, Hantavirus Infections immunology, Hantavirus Infections prevention & control, Hepatitis B Core Antigens genetics, Humans, Molecular Sequence Data, Plasmids genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Vaccines, Synthetic genetics, Vaccines, Synthetic pharmacology, Viral Vaccines genetics, Viral Vaccines pharmacology, Orthohantavirus immunology, Hepatitis B Core Antigens immunology
- Abstract
Virus-like particles generated by the heterologous expression of virus structural proteins are able to potentiate the immunogenicity of foreign epitopes presented on their surface. In recent years epitopes of various origin have been inserted into the core antigen of hepatitis B virus (HBV) allowing the formation of chimaeric HBV core particles. Chimaeric core particles carrying the 45 N-terminal amino acids of the Puumala hantavirus nucleocapsid protein induced protective immunity in bank voles, the natural host of this hantavirus. Particles applied in the absence of adjuvant are still immunogenic and partially protective in bank voles. Although a C-terminally truncated core antigen of HBV (HBcAg delta) tolerates the insertion of extended foreign sequences, for the construction of multivalent vaccines the limited insertion capacity is still a critical factor. Recently, we have described a new system for generating HBV 'mosaic particles' in an Escherichia coli suppressor strain based on a readthrough mechanism on a stop linker located in front of the insert. Those mosaic particles are built up by both HBcAg delta and the HBcAg delta/Puumala nucleocapsid readthrough protein. The particles formed presented the 114 amino acid (aa) long hantavirus sequence, at least in part, on their surface and induced antibodies against the hantavirus sequence in bank voles. Variants of the stop linker still allowed the formation of mosaic particles demonstrating that stop codon suppression alone is sufficient for the packaging of longer foreign sequences in mosaic particles. Another approach to increase the insertion capacity is based on the simultaneous insertion of different Puumala nucleocapsid protein sequences (aa 1-45 and aa 75-119) into two different positions (aa 78 and behind aa 144) of a single HBcAg molecule. The data presented are of high relevance for the generation of multivalent vaccines requiring a high insertion capacity for foreign sequences. more...
- Published
- 1999
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39. Isolation and molecular characterization of a novel cytopathogenic paramyxovirus from tree shrews.
- Author
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Tidona CA, Kurz HW, Gelderblom HR, and Darai G
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cell Line, Cloning, Molecular, Conserved Sequence, Cytopathogenic Effect, Viral, DNA, Complementary, DNA, Viral, Genes, Viral, Humans, Molecular Sequence Data, Phylogeny, Protein Sorting Signals, Rabbits, Respirovirus classification, Respirovirus isolation & purification, Respirovirus pathogenicity, Respirovirus Infections virology, Sequence Analysis, DNA, Transcription, Genetic, Viral Proteins genetics, Respirovirus genetics, Respirovirus Infections veterinary, Tupaia virology
- Abstract
A cytopathic infectious agent was isolated from the kidneys of an apparently healthy tree shrew (Tupaia belangeri) that had been captured in the area around Bangkok. The infectivity was propagated in Tupaia fibroblast and kidney cell cultures. Paramyxovirus-like pleomorphic enveloped particles and helical nucleocapsids were observed by electron microscopy and accordingly the infectious agent was termed Tupaia paramyxovirus (TPMV). However, no serological cross-reactions were detected between TPMV and known paramyxoviruses. For the molecular characterization of TPMV an experimental strategy that allows the random-primed synthesis of relatively large cDNA molecules from viral genomic RNA was applied. Nucleotide sequence analysis of a TPMV-specific cDNA fragment (1544 bp) revealed two nonoverlapping partial open reading frames corresponding to paramyxoviral N and P transcription units. Using modified rapid amplification of cDNA ends techniques, a substantial contiguous portion of the viral genome (4065 nt) was elucidated including the complete N and P/V/C genes. The coding strategy of TPMV as well as significant amino acid sequence homologies clearly indicates an evolutionary relationship between TPMV and members of the genus Morbillivirus. Highest homologies were detected between TPMV and Hendra virus (equine morbillivirus), which recently emerged in Australia, causing outbreaks of fatal respiratory and neurological disease in horses and humans., (Copyright 1999 Academic Press.) more...
- Published
- 1999
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40. Diagnostic electron microscopy is still a timely and rewarding method.
- Author
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Biel SS and Gelderblom HR
- Subjects
- Animals, Humans, Microscopy, Electron methods, Time Factors, Microscopy, Electron trends, Virus Diseases diagnosis
- Abstract
Background: Parallel to its technical development starting in the 1930s, electron microscopy (EM) became an important tool in basic and clinical virology. First utilized in the rapid diagnosis of smallpox, it developed to a diagnostic routine in the early 1960s using the negative staining technique. EM was applied to infected cell-cultures and also to 'dirty' specimens including urine, feces, vesicle fluid, liquor. With the implementation of molecular biological and genetic techniques, the use of diagnostic EM decreased., Objectives: (1) To give a perspective on future indications and possible uses by discussing the past and the present of diagnostic EM, (2) To describe the system of External Quality Assessment on EM virus diagnosis (EQA-EMV) established in 1994 by our laboratory and its achievements., Study Design: EQA-EMV is run to evaluate, to confirm and to improve the quality of diagnostic EM. Two different types of specimen are sent out: (1) prepared grids to assess and train the diagnostic skills of the participants, (2) stabilized virus particle suspensions to assess preparation efficiency., Results: Diagnostic EM differs from other diagnostic tests in its rapidity and its undirected 'open view'. To emphasize these advantages, the indications for diagnostic EM are discussed, fundamental for a continuing future adaptation. Besides appropriate techniques, quality control measures are required to achieve and keep high diagnostic standards. The results from 6 years of EQA-EMV are presented., Conclusions: In the history of diagnostic EM in virology, a change in use has been seen. Starting in the 1990s and coincident with the broad introduction of 'modern' diagnostic techniques, the number of EM diagnostic labs has decreased considerably--in spite of the obvious advantages of this technique. To guarantee the continuing performance of diagnostic EM in the future. EQA runs have to be performed as with other techniques in the diagnostic armament. The growing number of participants and participating countries indicates an interest in as well as a need for this program. more...
- Published
- 1999
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41. HBV core particles allow the insertion and surface exposure of the entire potentially protective region of Puumala hantavirus nucleocapsid protein.
- Author
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Koletzki D, Biel SS, Meisel H, Nugel E, Gelderblom HR, Krüger DH, and Ulrich R
- Subjects
- Animals, Binding Sites, Hepatitis B Core Antigens genetics, Humans, Nucleocapsid genetics, Nucleocapsid Proteins, Rabbits, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins immunology, Virion, Orthohantavirus immunology, Hepatitis B Core Antigens immunology, Hepatitis B virus immunology, Nucleocapsid immunology
- Abstract
Core particles of the hepatitis B virus (HBV) potentiate the immune response against foreign epitopes presented on their surface. Potential insertion sites in the monomeric subunit of the HBV core protein were previously identified at the N- and C-terminus and in the immunodominant c/e1 region. In a C-terminally truncated core protein these sites were used to introduce the entire 120 amino acid (aa)-long potentially immunoprotective region of the hantavirus (serotype Puumala) nucleocapsid protein. The N- and C-terminal fusion products were unable to form core-like particles in detectable amounts. However, a suppressable stop codon located between the HBV core and the C-terminally fused hantavirus sequence restored the ability to form particles ('mosaic particles'); in contrast to the C-terminal fusion product the mosaic construct allowed the formation of particles built up by the core protein itself and the HBV core-Puumala nucleocapsid-readthrough protein. The mosaic particles exposed the 120 aa region of the PUU nucleocapsid protein on their surface as demonstrated by ELISA and immuno electron microscopy applying different monoclonal antibodies. Insertion of the hantaviral sequence into the c/e1 region not only allowed the formation of chimeric particles, but again the surface accessibility of the sequence. HBV core antigenicity itself was, however, reduced in the particles carrying insertions in the c/e1 region, probably due to a masking effect of the 120 aa long insert. more...
- Published
- 1999
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- View/download PDF
42. Characterization of potential insertion sites in the core antigen of hepatitis B virus by the use of a short-sized model epitope.
- Author
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Lachmann S, Meisel H, Muselmann C, Koletzki D, Gelderblom HR, Borisova G, Krüger DH, Pumpens P, and Ulrich R
- Subjects
- Blotting, Western, Electrophoresis, Polyacrylamide Gel, Epitopes genetics, Genetic Vectors, Recombinant Proteins biosynthesis, Epitopes immunology, Hepatitis B Core Antigens genetics, Hepatitis B virus immunology, Transformation, Genetic
- Abstract
Core particles of hepatitis B virus (HBV) are able to improve the immunogenicity of foreign sequences exposed on the particle surface. The insertion site in the core antigen of HBV (HBcAg) determines the surface presentation and thus the immunogenicity of the foreign sequence. For direct comparison of the value of potential insertion sites in the core antigen, we constructed vectors allowing insertions of a model marker epitope DPAFR. This epitope was inserted at the N-terminus, the c/e1 loop, behind amino acid (aa) 144 and behind aa 183 (DPAF only). In addition, we generated a mosaic construct allowing the co-expression of HBcAg and a HBcAg/DPAFR fusion protein due to a suppressor tRNA-mediated readthrough mechanism. All 6 constructs allowed the formation of chimaeric or mosaic core-like particles. Western blot analyses and a direct ELISA demonstrated the presence of the DPAFR sequence in the chimaeric and mosaic particles. Competitive ELISA and immune electron-microscopic data suggested the c/e1 loop as the insertion site of choice for presenting foreign sequences on the surface of chimaeric HBV core particles. However, the N-terminal fusion also allowed partial surface exposure of the DPAFR motif. In contrast, in particles of constructs carrying the DPAFR insert at aa position 144 or 183, respectively, the epitope seemed not to be surface accessible. more...
- Published
- 1999
- Full Text
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43. Bacteriophage diversity in the North Sea.
- Author
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Wichels A, Biel SS, Gelderblom HR, Brinkhoff T, Muyzer G, and Schütt C
- Subjects
- Caudovirales isolation & purification, Caudovirales ultrastructure, Oceans and Seas, Phylogeny, Polymerase Chain Reaction methods, RNA, Ribosomal, 16S genetics, Bacteria virology, Caudovirales classification, Seawater virology
- Abstract
In recent years interest in bacteriophages in aquatic environments has increased. Electron microscopy studies have revealed high numbers of phage particles (10(4) to 10(7) particles per ml) in the marine environment. However, the ecological role of these bacteriophages is still unknown, and the role of the phages in the control of bacterioplankton by lysis and the potential for gene transfer are disputed. Even the basic questions of the genetic relationships of the phages and the diversity of phage-host systems in aquatic environments have not been answered. We investigated the diversity of 22 phage-host systems after 85 phages were collected at one station near a German island, Helgoland, located in the North Sea. The relationships among the phages were determined by electron microscopy, DNA-DNA hybridization, and host range studies. On the basis of morphology, 11 phages were assigned to the virus family Myoviridae, 7 phages were assigned to the family Siphoviridae, and 4 phages were assigned to the family Podoviridae. DNA-DNA hybridization confirmed that there was no DNA homology between phages belonging to different families. We found that the 22 marine bacteriophages belonged to 13 different species. The host bacteria were differentiated by morphological and physiological tests and by 16S ribosomal DNA sequencing. All of the bacteria were gram negative, facultatively anaerobic, motile, and coccoid. The 16S rRNA sequences of the bacteria exhibited high levels of similarity (98 to 99%) with the sequences of organisms belonging to the genus Pseudoalteromonas, which belongs to the gamma subdivision of the class Proteobacteria. more...
- Published
- 1998
- Full Text
- View/download PDF
44. Effects of IFN alpha on late stages of HIV-1 replication cycle.
- Author
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Dianzani F, Castilletti C, Gentile M, Gelderblom HR, Frezza F, and Capobianchi MR
- Subjects
- Gene Products, gag metabolism, Genome, Viral, HIV Core Protein p24 metabolism, HIV Envelope Protein gp120 metabolism, HIV-1 physiology, Humans, Protein Precursors metabolism, Protein Processing, Post-Translational, Time Factors, U937 Cells, Virus Assembly, Antiviral Agents pharmacology, HIV-1 drug effects, Interferon-alpha pharmacology, Virus Replication
- Abstract
IFN alpha causes a modest reduction of HIV-1 expression in chronically infected monocytoid U937 cells. However, the ratio between cell-associated and shed viral p24 antigen is altered, being the cell-associated fraction dose-dependently enhanced by IFN. Furthermore, a significant decrease of infectivity of both cell-associated and shed material is observed. Transmission electron microscopy of IFN-treated cells revealed virus assembly being strongly inhibited, with the production of morphologically altered (tear-drop shaped) virus particles. Proteolytic processing of gag proteins appeared to be normal in IFN-treated cultures. However, virions shed from IFN-treated cells showed a markedly reduced incorporation of virus-specific gp120 and cell-derived ICAM-1 by the virus envelope. Additionally, these particles showed a significantly decreased ability to become bound to CD4+ target cells, accounting for, at least in part, the observed decrease of infectivity. Taken together, the data suggest that, in chronically infected cells, IFN alpha can affect late stages of HIV-1 replication, by inhibiting virus assembly and release, and by reducing the infectivity of shed virions. The latter effect seems to be due, at least in part, to altered incorporation of surface glycoproteins and defective particle formation. The relationship between impaired gp120 incorporation and altered morphogenesis of HIV-1 virions is under investigation. more...
- Published
- 1998
- Full Text
- View/download PDF
45. Efficient HIV-1 replication can occur in the absence of the viral matrix protein.
- Author
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Reil H, Bukovsky AA, Gelderblom HR, and Göttlinger HG
- Subjects
- Base Sequence, Cell Line, Gene Products, env biosynthesis, Gene Products, vpr biosynthesis, Genes, env, Genetic Complementation Test, HIV Core Protein p24 biosynthesis, HIV Core Protein p24 metabolism, HIV-1 genetics, HIV-1 ultrastructure, HeLa Cells, Humans, Microscopy, Electron, Molecular Sequence Data, Mutagenesis, Site-Directed, Oligodeoxyribonucleotides, Proviruses physiology, Recombinant Proteins metabolism, Transfection, Virion genetics, Virion physiology, Virion ultrastructure, vpr Gene Products, Human Immunodeficiency Virus, HIV-1 physiology, Viral Matrix Proteins metabolism, Virus Replication
- Abstract
Matrix (MA), a major structural protein of retroviruses, is thought to play a critical role in several steps of the HIV-1 replication cycle, including the plasma membrane targeting of Gag, the incorporation of envelope (Env) glycoproteins into nascent particles, and the nuclear import of the viral genome in non-dividing cells. We now show that the entire MA protein is dispensable for the incorporation of HIV-1 Env glycoproteins with a shortened cytoplasmic domain. Furthermore, efficient HIV-1 replication in the absence of up to 90% of MA was observed in a cell line in which the cytoplasmic domain of Env is not required. Additional compensatory changes in Gag permitted efficient virus replication even if all of MA was replaced by a heterologous membrane targeting signal. Viruses which lacked the globular domain of MA but retained its N-terminal myristyl anchor exhibited an increased ability to form both extracellular and intracellular virus particles, consistent with a myristyl switch model of Gag membrane targeting. Pseudotyped HIV-1 particles that lacked the structurally conserved globular head of MA efficiently infected macrophages, indicating that MA is dispensable for nuclear import in terminally differentiated cells. more...
- Published
- 1998
- Full Text
- View/download PDF
46. Chimaeric HBV core particles carrying a defined segment of Puumala hantavirus nucleocapsid protein evoke protective immunity in an animal model.
- Author
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Ulrich R, Lundkvist A, Meisel H, Koletzki D, Sjölander KB, Gelderblom HR, Borisova G, Schnitzler P, Darai G, and Krüger DH
- Subjects
- Animals, Arvicolinae, Hantavirus Infections immunology, Viral Vaccines immunology, Orthohantavirus immunology, Hantavirus Infections prevention & control, Hepatitis B Core Antigens immunology, Nucleocapsid immunology, Recombinant Fusion Proteins immunology, Viral Vaccines administration & dosage
- Abstract
Hantaviruses are rodent-born agents which are pathogenic in humans causing haemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. To induce a protective immunity against a European hantavirus (Puumala) we constructed chimaeric hepatitis B virus (HBV) core particles carrying defined fragments of the Puumala virus nucleocapsid protein. After immunisation of bank voles, the natural host of Puumala virus, with core particles possessing an insertion of the N-terminal part of Puumala virus nucleocapsid protein, four of five animals were protected against subsequent virus challenge. The results show that the major protective region of the nucleocapsid protein is located between amino acids 1 and 45 and that chimaeric HBV core-like particles are useful carriers of foreign protective epitopes. more...
- Published
- 1998
- Full Text
- View/download PDF
47. Identification of the UL56 protein of herpes simplex virus type 1 within the virion by immuno electron microscopy.
- Author
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Kehm R, Gelderblom HR, and Darai G
- Subjects
- Herpesvirus 1, Human chemistry, Immunoblotting, Microscopy, Immunoelectron, Virion chemistry, Herpesvirus 1, Human ultrastructure, Viral Proteins analysis, Virion ultrastructure
- Abstract
Recently the UL56 protein of herpes simplex virus type 1 (HSV-1) was shown to be associated with the virion of HSV-1 as determined by Western blot analysis. The detection of the UL56 protein in infected cells and its association with virions of HSV-1 is of particular importance, pointing to a possible involvement of UL56 protein in virus-host interactions. In order to investigate the properties of the UL56 protein further immuno-localization was performed using rabbit hyperimmune serum against fusion recombinant UL56 protein and purified virions of HSV-1 strain F. The UL56 protein was detected in the HSV-1 virions by immuno gold negative staining. more...
- Published
- 1998
- Full Text
- View/download PDF
48. Monocyte-derived dendritic cells represent a transient stage of differentiation in the myeloid lineage.
- Author
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Häusser G, Ludewig B, Gelderblom HR, Tsunetsugu-Yokota Y, Akagawa K, and Meyerhans A
- Subjects
- Antigen Presentation, Antigens, CD biosynthesis, CD4-Positive T-Lymphocytes immunology, CD40 Ligand, Cell Adhesion, Cell Differentiation drug effects, Cell Lineage, Granulocyte-Macrophage Colony-Stimulating Factor pharmacology, HLA Antigens biosynthesis, Humans, Langerhans Cells immunology, Lymphocyte Activation, Lymphocyte Culture Test, Mixed, Macrophage Colony-Stimulating Factor pharmacology, Macrophages cytology, Membrane Glycoproteins pharmacology, Monocytes cytology, Tumor Necrosis Factor-alpha pharmacology, Dendritic Cells cytology, Monocytes drug effects
- Abstract
Cultivation of human peripheral blood monocytes with granulocyte/macrophage colony stimulating factor (GM-CSF) and IL-4 facilitates generation of strongly antigen-presenting dendritic cells (DC). These monocyte-derived DC (mdDC) were used here to further delineate differentiation pathways in the myeloid lineage. Incubation of mdDC with TNF or soluble CD40L led to enhanced MHC and accessory surface antigen expression with significantly elevated T cell stimulatory activity, indicative of DC maturation. In contrast, after cytokine withdrawal or incubation with M-CSF, mdDC differentiated to macrophages. Cells became adherent, monocyte/macrophage surface markers were upregulated, and MHC and accessory surface proteins were downregulated. Furthermore, the multilaminar MHC class II compartments (MIIC) were lost and the T cell stimulating capacity largely diminished. Thus, mdDC show a high developmental plasticity by retaining their ability to become macrophages or to continue their differentiation towards mature DC. more...
- Published
- 1997
- Full Text
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49. Mosaic hepatitis B virus core particles allow insertion of extended foreign protein segments.
- Author
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Koletzki D, Zankl A, Gelderblom HR, Meisel H, Dislers A, Borisova G, Pumpens P, Krüger DH, and Ulrich R
- Subjects
- Amino Acid Sequence, Base Sequence, Drug Design, Escherichia coli, Hepatitis B Core Antigens ultrastructure, Microscopy, Electron, Mosaicism, Nucleocapsid ultrastructure, Plasmids, Recombinant Fusion Proteins ultrastructure, Sequence Deletion, Vaccines, Synthetic, Viral Vaccines, Cloning, Molecular methods, Orthohantavirus genetics, Hepatitis B Core Antigens biosynthesis, Hepatitis B virus genetics, Mutagenesis, Insertional methods, Nucleocapsid biosynthesis, Recombinant Fusion Proteins biosynthesis
- Abstract
Because of its particular immunological properties, the core protein of hepatitis B virus (HBcAg) has become one of the favoured 'virus-like particles' for use as a carrier of foreign epitopes. A new strategy to construct core particles presenting extended foreign protein segments was established based on the introduction of a linker containing a translational stop codon between sequences encoding a C-terminally truncated HBcAg (HBcAg delta) and a foreign protein sequence. Expression in an Escherichia coli suppressor strain allowed the simultaneous synthesis of both HBcAg delta and a read-through fusion protein containing a part of the hantavirus nucleocapsid protein. After purification, the presence of core-like mosaic particles with HBc and hantavirus antigenicity was demonstrated by electron microscopy and immunological tests. This strategy of partial stop codon suppression should improve the use of HBcAg as a carrier of foreign epitopes by allowing insertion of long foreign sequences into particle-forming proteins. The resulting mosaic particles should be of general interest for further vaccine developments. more...
- Published
- 1997
- Full Text
- View/download PDF
50. Cell membrane vesicles are a major contaminant of gradient-enriched human immunodeficiency virus type-1 preparations.
- Author
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Gluschankof P, Mondor I, Gelderblom HR, and Sattentau QJ
- Subjects
- Cell Membrane metabolism, Humans, Tumor Cells, Cultured, Antigens, CD metabolism, HIV Core Protein p24 metabolism, HIV-1 isolation & purification, HLA-DR Antigens metabolism
- Abstract
During preliminary experiments to establish the proportion of virus-coded p24 protein to virus membrane-associated HLA-DR in gradient-enriched HIV-1 preparations, we became aware of a large variability between experiments. In order to determine whether HLA-DR-containing cellular material was contaminating the virus preparations, we carried out enrichment by gradient centrifugation of clarified supernatants from noninfected cells and tested this material for HLA-DR content. We found that, independently of the cell type used, gradient enrichment resulted in the isolation of large quantities of HLA-DR-containing material which banded at a density overlapping that of infectious HIV. Electron microscopy of gradient-enriched preparations from supernatants of virus-infected cells revealed an excess of vesicles with a size range of about 50-500 nm, as opposed to a minor population of virus particles of about 100 nm. Electron micrographs of infected cells showed polarized vesiculation of the cell membrane, and virus budding was frequently colocalized with nonviral membrane vesiculation. Analysis of the cellular molecules present in the fractions containing virus or exclusively cellular material demonstrated that virus and cellular vesicles share several cellular antigens, with the exception of CD43 and CD63, found mainly at the virus surface, and HLA-DQ, which was found only in the cellular vesicles. more...
- Published
- 1997
- Full Text
- View/download PDF
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