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Biomedical subjects

B Ehlers

Publications and source records attributed to B Ehlers.

At least 19 recordsLinked to original sources

The porcine lymphotropic herpesvirus 1 encodes functional regulators of gene expression.

The porcine lymphotropic herpesviruses (PLHV) are discussed as possible risk factors in xenotransplantation because of the high prevalence of PLHV-1, PLHV-2 and PLHV-3 in pig populations world-wide and the fact that PLHV-1 has been found to be associated with porcine post-transplant lymphoproliferative disease. To provide structural and functional knowledge on the PLHV immediate-early (IE) transactivator genes, the central regions of the PLHV genomes were characterized by genome walking, sequence and splicing analysis. Three spliced genes were identified (ORF50, ORFA6/BZLF1(h), ORF57) encoding putative IE transactivators, homologous to (i) ORF50 and BRLF1/Rta, (ii) K8/K-bZIP and BZLF1/Zta and (iii) ORF57 and BMLF1 of HHV-8 and EBV, respectively. Expressed as myc-tag or HA-tag fusion proteins, they were located to the cellular nucleus. In reporter gene assays, several PLHV-promoters were mainly activated by PLHV-1 ORF50, to a lower level by PLHV-1 ORFA6/BZLF1(h) and not by PLHV-1 ORF57. However, the ORF57-encoded protein acted synergistically on ORF50-mediated activation.

Animals↗

Fatal poxvirus outbreak in a colony of New World monkeys.

An epizootic infection was observed in a colony of 80 New World monkeys consisting of various species including a group of marmosets and Saguinus species. During the summer and autumn of 2002, 30 animals died of unknown diseases. Six animals were sent to the German Primate Center for investigation of the cause of death. A complete pathologic and histologic investigation was carried out. The animals exhibited erosive-ulcerative lesions of the oral mucous membranes. Advanced stages of the disease were characterised by hemorrhagic lesions on the skin distributed randomly over the body, but principally on the face, scrotal region, soles, and palms. Electron microscopy revealed virus particles with orthopox-like morphology within intracytoplasmic inclusions in epithelial cells. The DNA samples from various tissues were analyzed by use of a set of orthopox virus-specific, real-time polymerase chain reaction assays. Amplification products were sequenced to define the virus more precisely. Sequencing confirmed the presence of an orthopox virus. Sequence data indicated that all six animals were infected with the same virus. Propagation of the virus on Vero cells resulted in a rapidly progressive cytopathogenic effect. Preliminary phylogenetic analyses of two genes revealed closest homology to cowpox viruses. The origin of this poxvirus outbreak remains unexplained, and the strain and genus of the virus need to be determined in detail.

Animals↗

[Porcine malignant catarrhal fever: diagnostic findings and first detection of the pathogenic agent in diseased swine in Switzerland].

For the first time Ovine Herpesvirus 2 (OvHV-2) was identified in Swiss pigs as the causative agent of Porcine Malignant Catarrhal Fever (MCF). Diseased animals from two farms were observed to show weakness, anorexia, fever up to 41 degrees C, and neurological symptoms, i.e. ataxia, convulsions and hyperesthesia, erosion on the snout and in the oral and nasal mucosa, as well as multiple skin lesions. Histopathological findings included severe non-purulent inflammation with mononuclear cell infiltration in several organs. Most dominant were meningo-encephalitis, disseminated nephritis as well as purulent catarrhalic bronchopneumonia. The findings were quite reminiscent of the lesions due to MCF in cattle and give therefore substantial proof to use Porcine Malignant Catarrhal Fever as the term for the disease. Identification of the causative agent was done with a quantitative PCR specific for OvHV-2. Different tissues from diseased animals were positive. Furthermore, one animal which had been ill for more than five days tested positive for antibodies against an epitope conserved among MCF viruses. Serum samples from diseased animals reacted negative towards Classical Swine Fever- and Pseudorabies virus antigen. A weakly positive reaction against porcine enterovirus type I argued against the involvement of enteroviruses in the observed disease. Moreover, by means of different conventional PCRs, we detected the newly discovered porcine lymphotropic herpesviruses for the first time in Switzerland and could at the same time exclude their involvement in Porcine Malignant Catarrhal Fever.

Animals↗

Detection and multigenic characterization of a novel gammaherpesvirus in goats.

Evidence for the existence of a caprine gammaherpesvirus was obtained by analysis of peripheral blood leucocytes of goats with PCR assays that target the herpesvirus genes encoding the glycoprotein B (gB), the DNA polymerase (DPOL) and the terminase (TERM) with degenerate and deoxyinosine-substituted primers. A contiguous 3.6 kbp sequence extending from the gB to the DPOL gene was then determined with specific primers. All sequences (gB, DPOL and TERM) showed a close relationship with the corresponding genes of the Gammaherpesvirinae. Alignment of amino acid sequences revealed a particularly high percentage of identity with the ovine herpesvirus type 2 (>83%), followed by the alcelaphine herpesvirus 1 (>76%) and the bovine lymphotropic herpesvirus (>61%). Phylogenetic analyses confirmed these relationships. The putative novel goat herpesvirus from which these sequences originate was tentatively designated caprine herpesvirus 2. This virus is the first gammaherpesvirus recognized in goats.

Animals↗

Identification and sequence analysis of the glycoprotein B gene of porcine cytomegalovirus.

Porcine cytomegalovirus (PCMV) is one of the pathogens that should be eliminated from pigs intended for use as organ donors in xenotransplantation. For this purpose, reliable diagnostic test systems are needed. To provide a basis for this goal and to analyse the evolutionary relationships of PCMV within the herpesvirus family, the putative glycoprotein B (gB) gene of PCMV was identified by assuming gene colinearity and a relative conservation of nucleotide sequences in comparison with closely related herpesviruses. Using this approach the complete nucleotide sequence of the PCMV gB gene was determined. A protein of 860 amino acids was deduced and a putative cleavage site, conserved cysteine residues, as well as potential N-terminal glycosylation motifs were identified. In a comparison of PCMV gB with the corresponding region of other herpesviruses, the highest identities were found with human herpesviruses 6 and 7 (HHV-6 and 7; 43.4% and 42.6%, respectively). Also in phylogenetic analysis, the PCMV gB clustered with HHV-6 and HHV-7. Between the complete gB sequences of five different PCMV strains and isolates from the United Kingdom, Germany, Spain, Japan and Sweden, differences of 3.4% were found, indicating a considerable intra-species variation. The characterisation of the protein deduced from the identified gene provides further evidence that this is indeed the gB gene of PCMV and provides important taxonomical information regarding PCMV. The identification of the gB gene should facilitate the development of sensitive and robust diagnostic methods for the PCMV screening of pigs.

Amino Acid Sequence↗

Genetic and ultrastructural characterization of a European isolate of the fatal endotheliotropic elephant herpesvirus.

A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (ElHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, ElHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, ElHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant.

Animals↗

Genome sequence of bovine herpesvirus 4, a bovine Rhadinovirus, and identification of an origin of DNA replication.

Bovine herpesvirus 4 (BoHV-4) is a gammaherpesvirus of cattle. The complete long unique coding region (LUR) of BoHV-4 strain 66-p-347 was determined by a shotgun approach. Together with the previously published noncoding terminal repeats, the entire genome sequence of BoHV-4 is now available. The LUR consists of 108,873 bp with an overall G+C content of 41.4%. At least 79 open reading frames (ORFs) are present in this coding region, 17 of them unique to BoHV-4. In contrast to herpesvirus saimiri and human herpesvirus 8, BoHV-4 has a reduced set of ORFs homologous to cellular genes. Gene arrangement as well as phylogenetic analysis confirmed that BoHV-4 is a member of the genus Rhadinovirus. In addition, an origin of replication (ori) in the genome of BoHV-4 was identified by DpnI assays. A minimum of 1.69 kbp located between ORFs 69 and 71 was sufficient to act as a cis signal for replication.

Animals↗

Cloning and sequencing of columbid circovirus (coCV), a new circovirus from pigeons.

The complete nucleotide sequence of columbid circovirus (CoCV) isolated from pigeons is described. CoCV was amplified using a consensus primer PCR approach directed against conserved sequences within the rep genes of vertebrate circoviruses. The genome of CoCV is circular and 2037 nt in size. It displays 55% homology to the genome of psittacine beak and feather disease virus and is more distantly related (< 40% homology) to porcine circovirus type 1 and 2. Two major open reading frames were identified, encoding the replicase and the putative capsid protein of CoCV. A region similar to the origin of replication of other circoviruses was found: it encompasses a stem-loop structure with the nonamer 5'-TAGTATTAC, conserved in circo-, nano- and geminiviruses. Phylogenetic analyses suggest classification of CoCV as member of the genus Circovirus of the virus family Circoviridae.

Amino Acid Sequence↗

Characterization of the DNA polymerase loci of porcine cytomegaloviruses from diverse geographic origins.

Porcine cytomegalovirus (PCMV) is an undesired pathogen in pigs intended for use as organ donors in xenotransplantation. In the present work, we characterized the first set of genes of PCMV. From a German isolate, the DNA polymerase (DPOL) locus was amplified and two complete open reading frames (ORF) as well as two partial ORFs including the complete DPOL gene and the 3'-end of the glycoprotein gB gene were sequenced. The deduced amino acid sequences showed the highest identities with the respective proteins of the betaherpesviruses, in particular those (ORFs 36-39) of the human herpesviruses 6 and 7 (HHV-6 and -7). In phylogenetic analysis, PCMV clustered also with HHV-6 and HHV-7. On this basis, PCMV could be firmly classified to the Betaherpesvirinae and tentatively assigned to the genus Roseolovirus. In addition to the German isolate, the DPOL gene was analysed from a British and a Japanese strain as well as a Spanish isolate. Differences of 0.4 to 1% were found on the nucleotide and the amino acid level. On the basis of the conserved regions, primer pairs were selected for PCR which detected PCMV in blood and tissue samples from four European countries. Therefore, these are the first nucleic acid-based test systems which were shown to universally detect PCMV. The application of these assays to organs of domestic pigs from Germany revealed a PCMV prevalence of > 50%.

Animals↗

Detection of new DNA polymerase genes of known and potentially novel herpesviruses by PCR with degenerate and deoxyinosine-substituted primers.

A consensus primer PCR approach was used to (i) investigate the presence of herpesviruses in wild and zoo equids (zebra, wild ass, tapir) and to (ii) study the genetic relationship of the herpesvirus of pigeons (columbid herpesvirus 1) to other herpesvirus species. The PCR assay, based on degenerate primers targeting highly conserved regions of the DNA polymerase gene of herpesviruses, was modified by using a mixture of degenerate and deoxyinosine-substituted primers. The applicability of the modification was validated by amplification of published DNA polymerase genes of 16 herpesvirus species and of the previously uncharacterized DNA polymerase genes of equine herpesvirus 3 (EHV-3) and equine herpesvirus 5 (EHV-5). The modified assay was then used for partial amplification of the polymerase of columbid herpesvirus 1 which is presently classified as a beta-herpesvirus based on biological criteria. Sequence analysis of amplicons obtained from four different viral strains revealed a close relationship of columbid herpesvirus 1 to members of the subfamily Alphaherpesvirinae, especially to Marek's disease herpesvirus. This was confirmed by characterization of additional 1.6kb of the columbid herpesvirus 1 polymerase. Consensus PCR analysis of blood samples from zebras, a wild ass and a tapir revealed amplicons showing high percentages ( > 50%) of sequence identity to DNA polymerases of gamma-herpesviruses. In particular, the zebra and the wild ass sequence were closely related to each other and to the polymerases of the equine gamma-herpesviruses EHV-2 and EHV-5 with sequence identities of > 80%. This is a first indication that novel gamma-herpesviruses are present in wild and zoo equids.

Amino Acid Sequence↗

Bovine herpesvirus type 2 is closely related to the primate alphaherpesviruses.

Bovine herpesvirus type 2 (BoHV-2), also known as bovine mammillitis virus, is classified in the Family Herpesviridae, Subfamily Alphaherpesvirinae, and Genus Simplexvirus along with herpes simplex viruses type 1 and 2 (HSV-1 and HSV-2) and other primate simplexviruses on the basis of similarities in 4 genes within the 15 kb U(L) 23-29 cluster. This could be explained either by a global similarity or a recombination event that brought primate herpesviral sequences into a bovine virus. Our sequences for DNA polymerase (U(L)30), a large gene adjacent to the previously identified conserved cluster, and glycoprotein G (U(S)4), a gene as distant from the cluster as possible on the circularized genome, confirm the close relationship between BoHV-2 and the primate simplexviruses, and argue for a global similarity and probably a close evolutionary relationship. Thus one can speculate that BoHV-2 may represent a greater hazard to humans than has been appreciated previously.

Alphaherpesvirinae↗

Detection of two novel porcine herpesviruses with high similarity to gammaherpesviruses.

Evidence for the existence of porcine gammaherpesviruses was obtained by PCR and sequence analysis. Initially, samples of peripheral blood mononuclear cells (PBMC), spleens, lungs, kidneys and livers of pigs from Germany and Spain were tested with a PCR assay which targets conserved regions of the herpesvirus DNA polymerase gene with degenerate and deoxyinosine-substituted primers. Amplicons of identical sequence were obtained from one spleen and two PBMC samples. This sequence showed a high percentage of identity with the DNA polymerase genes of herpesviruses of the oncogenic subfamily Gammaherpesvirinae. Alignment of amino acid sequences showed the highest identity values with bovine gammaherpesviruses, namely alcelaphine herpesvirus type 1 (68%), ovine herpesvirus type 2 (68%) and bovine lymphotropic herpesvirus (67%). Comparison with pseudorabies virus and porcine cytomegalovirus, which are the only porcine herpesvirus species presently known, showed values of only 41%. PCR analysis of PBMC (n = 39) and spleen (n = 19) samples from German pigs, using primers specific for the novel sequence, revealed a prevalence of 87 and 95%, respectively. In this analysis, three out of eight spleen samples from Spanish pigs were also positive. Subsequent sequencing of the amplicons revealed the presence of two closely related gammaherpesvirus sequences, differing from each other by 8% at the amino acid level. The putative novel porcine herpesviruses, from which these sequences originated, were tentatively designated porcine lymphotropic herpesvirus type 1 and type 2 (PLHV-1 and PLHV-2). When using pig organs for xenotransplantation, the presence of these viruses has to be considered.

Amino Acid Sequence↗

[Elephant herpes virus--a problem for breeding and housing of elephants].

Herpesvirus infections which take a fatal turn on African elephants as well as on Asian elephants seem to occur increasingly not only in the USA but also in European stocks. The endotheliotropic herpesvirus causes a rapidly progressing and severe disease which makes any therapeutical effort unsuccessful and finally results in death of the animal, especially in young Asian elephants. As all attempts to culture the virus failed up to now, molecular biological procedures have to be used more often for diagnostical purpose together with the common methods of pathology, virology, and electronmicroscopical evaluation. This is a report on the case of 'KIBA', an eleven year old male elephant at the Zoological Garden Berlin, infected with the endotheliotropic elephants herpesvirus. 'KIBA' was born at the Zoo in Houston, Texas, and raised within his herd. Upon arriving in Berlin in November 1997 he adapted to the new premises and climate and new social circumstances without any problems. In June 1998 he already serviced three females of his new herd several times. In August 1998 he died after passing a peracute progression of the disease after residenting in Berlin for only 9 months. The dissection of the animal revealed some evidence on an agent damaging the endothelium. Major signs indicating this agent were bleedings in several serous membranes, mucosa and on the the right atrium, as well as other parts of the myocardium. Furthermore there have been ulcerations at various localisations of the whole digestive tract. Slightly basophilic intranuclear inclusion bodies have been found histologically in endothelial cells of different organ samples. An examination of altered organ-material by electronmicroscopy made some herpesvirus-like particles visible. A virological investigation first revealed evidence of giant cell formations with solitary basophilic intranuclear inclusion bodies in different cell cultures, however, without any distinct cytopathogenic effect. Supported by molecular biological procedures the infection of 'KIBA' could be verified as the elephants herpesvirus. By means of PCR and subsequent sequence analysis a DNA-sequence typical for the elephants herpesvirus could be obtained which showed an identity of 97% with the terminase sequence of the elephant herpesvirus described by American authors. The deduced amino acid-sequences were 100% identical. To the terminase of the human cytomegalovirus, the elephant sequence had an identity of 53% (similarity: 74%). Based on the cooperation of ILAT, Institute of Veterinary-Pathology/Free University Berlin, Robert-Koch-Institut Berlin, and Zoological Garden Berlin, the cause of 'KIBA's' death could be discovered immediately. Possible implications of this case especially on breeding and keeping elephants are discussed briefly.

Amino Acid Sequence↗

Efficacy of a low molecular weight heparin administered intravenously or subcutaneously in comparison with intravenous unfractionated heparin in the treatment of deep venous thrombosis. Certoparin-Study Group.

BACKGROUND: The main objective of the study presented was to test if thrombus regression can be improved by treatment with an intravenously or subcutaneously administered low molecular weight heparin (LMWH). Patients with acute deep vein thrombosis were randomly assigned to receive either intravenous UFH (131 patients), intravenous (i.v.) LMWH (128 patients), or 8000 IU of the same LMWH bid subcutaneously (s.c.) (128 patients). All patients were treated with heparin for 14 to 16 days. Vitamin-K-antagonist prophylaxis was started between Day 12 and Day 14 after enrollment into the study. METHODS: Phlebographies and perfusion/ventilation lung scans were performed at baseline and on Days 12 to 16. Primary endpoint of the study was a reduction of the phlebographic Marder score. Secondary endpoints were recurrent thrombosis and pulmonary embolism (PE), major and minor bleedings and the rate of PE at inclusion and at the end of the study assessed by ventilation/perfusion scans. RESULTS: The Marder score improved by at least 30% in 32.4% (95% CI: 22.6 ... 42.2) of the patients receiving UFH, in 34.0% (95% CI: 24.9 ... 44.0) receiving LMWH i.v. and in 42.6% (95% CI: 32.8 ... 52.8) treated with the low molecular weight heparin s.c. The difference between LMWH s.c. and UFH was 10.2% (95% CI: -3.7% ... +24.5%) (p = 0.11). PE with clinical signs confirmed by objective methods occurred in three patients of the UFH group, one of whom died and was not observed in patients of the i.v. or s.c. LMWH-groups. During the first 15 days no patient receiving UFH or i.v. LMWH, and one patient on s.c. LMWH had a recurrent thrombosis. Major bleedings were observed in four patients receiving i.v. UFH compared to nine patients on i.v. LMWH (one of these patients died) and one patient on s.c. LMWH. Perfusion ventilation lung scans were obtained from 287 patients at baseline and from 246 patients on Days 12-16. PE, defined according to PIOPED-criteria as intermediate or high probability scans, was observed in 38.0% of the patients entering the study and in 18.3% on Days 12 to 16. New asymptomatic PE occurred less frequently in the groups on LMWH (7.1%, 7.5%, respectively) than in the UFH-group (12.6%) (not significant). CONCLUSIONS: S.c. treatment with a LMWH (certoparin) (b.i.d.) is at least as effective as UFH i.v. The hypothesis of increased efficacy of subcutaneous LMWH in resolving venous thrombi will have to be confirmed by an independent study comparing s.c. LMWH with UFH. The i.v. continuous infusion of the LMWH for 12 to 16 days does not result in a higher venous re-opening rate than intravenous standard heparin.

Adolescent↗

[Epidemiological and prognostic data from 2054 patients of an internal medicine intensive care unit].

BACKGROUND: Computer-based data collection and objective gathering of degree of illness severity and risk of death with a prognostic scoring system make it possible to obtain, in addition to epidemiological and aetiological data, risk-related outcome values for patients in an intensive care unit. PATIENTS AND METHODS: All 2054 patients who during a 2-year period (1995-1996) had stayed in a medical intensive care unit (MICU) for more than 4 hours were studied prospectively. The simplified acute physiology score II (SAPS II), risk of death, duration of stay in the MICU and in the hospital, and death rates during MICU and hospital stay were determined. Mean and median values and histograms of the various parameters as well as the standardized mortality index (SMI: observed/ predicted death rate with 99% confidence limits) were calculated for each of the patients and certain defined subgroups (basic disease, age, risk). Receiver operating characteristics curves (discrimination) and calibration curves were obtained for SAPS II. RESULTS: Mean age for the cohort was 59.8 years, duration of stay in the MICU 3.1 days, in hospital 14.7 days, SAPS II was 30.3 points, death risk 0.17, death rate during ICU stay was 8.3%, during hospital stay 13.9% and the SMI 0.8% (0.74-0.88). Cardiac disease was the most common underlying condition (60%), while the small group of neurological conditions was remarkable for the high degree of severity and unfavourable prognosis. Both death rate and degree of disease severity increased with age. But the SMI was not significantly higher than 1.0 in both the elderly patients and the high-risk group of patients (on ventilator, renal replacement procedures, death risk > 0.5). CONCLUSIONS: Most patients in a MICU have underlying cardiac disease. Permanently available neurological consultation is essential. The high hospital death rate for elderly patients and those requiring respiratory support is a problem of disease severity, not of the quality of treatment. The risk of death is high on transfer to a general ward. Determination of the SMI is recommended for internal quality control in an ICU.

Adult↗

Macrolipasaemia in a patient with pancreas divisum and acute abdominal pain: a case report.

This study describes a rapid increase of serum lipase activity during an episode of severe abdominal pain in a female patient with macrolipasaemia and pancreas divisum. Serum lipase activity was persistently elevated in the patient with simultaneously low levels of amylase activity which only increased in association with endoscopic retrograde cholangiopancreatography. Polyethylene glycol precipitation experiments, as well as the results of exclusion chromatography, demonstrated that the high lipase activity was due to a macromolecule of approximately 200000 Da. Affinity chromatography and immunological investigations further indicated that the atypical time-course of lipase activity was not due to alterations of the patient's lipase, but due to lipase-binding autoantibodies resulting in the formation of immune complexes between lipase and immunoglobulin G lambda. Our results show that macrolipasaemia must be considered as a possible cause in patients with unexplained raised serum lipase activity.

Abdominal Pain↗

Serum levels of soluble CD23, but not soluble CD25, predict disease progression in early stage B-cell chronic lymphocytic leukemia.

Serum levels of the soluble forms of CD23 (sCD23) and CD25 (sCD25) were prospectively analyzed with respect to their prognostic relevance in early stage B-cell chronic lymphocytic leukemia (B-CLL). SCD23 and sCD25 levels were determined in 105 patients with newly diagnosed B-CLL (Binet stage A). In 93 of the patients, these levels were correlated with other already established indicators for risk of disease progression, including the histologic pattern of bone marrow infiltration, lymphocyte doubling time (LDT), and the serum level of thymidine kinase (TK). High serum levels of both sCD23 and of sCD25 were associated with a diffuse bone marrow infiltration, a LDT < or = 12 months, and elevated (>5 U/L) serum TK, respectively. Moreover, examination of the clinical course of 76 untreated patients showed that high levels of sCD23, but not of sCD25, at initial diagnosis were linked with disease progression. Furthermore, in a stepwise Cox regression model, high levels of sCD23 and a short LDT were shown to be strong predictors of progressive disease within the first year of disease presentation. Therefore, it appears to be justified to incorporate sCD23 levels into the risk profile of early stage B-CLL and to take them into account for stratification in risk-adapted treatment strategies.

Biomarkers, Tumor↗