Biomedical subjects

Thomas Juretzek

Publications and source records attributed to Thomas Juretzek.

5 recordsLinked to original sources

PubMed2004

A simple and rapid single-step multiplex RT-PCR to detect Norovirus, Astrovirus and Adenovirus in clinical stool samples.

A single-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay that detects and identifies Norovirus, Astrovirus and Adenovirus in clinical stool samples is described. Four hundred sixty stool samples were tested from patients with non-rotavirus acute gastroenteritis, that were either stored at -80 degrees C and tested retrospectively, or tested immediately after viral nucleic acid extraction in a prospective manner, including outbreaks of gastroenteritis that occurred in Germany during the winter of 2003. The multiplex RT-PCR was validated against simplex RT-PCR with published primers for Norovirus (JV12/JV13 and p289/p290) and Astrovirus (Mon340/348), and against simplex PCR for Adenovirus. In both retrospective and prospective settings, the multiplex RT-PCR was equally sensitive and specific in detecting non-rotavirus infections compared with simplex RT-PCR/PCR. The specificity of the multiplex RT-PCR was assessed by sequencing of the amplicons that showed high nucleotide identities to Norovirus genogroup I/1, I/4, II/2, or II/4 clades, as well as to Astrovirus serotypes 1, 2, 4, or 8. The multiplex RT-PCR was also more sensitive than Astrovirus and Norovirus antigen enzyme immunoassays (IDEIA, Dako), as well as Astrovirus isolation in cell culture. This novel multiplex RT-PCR is an attractive technique for the rapid, specific, and cost-effective laboratory diagnosis of non-rotavirus acute gastroenteritis.

Adenoviridae↗

PubMed2004

Foamy virus integration.

It had been suggested that during integration of spumaretroviruses (foamy viruses) the right (U5) end of the cDNA is processed, while the left (U3) remains uncleaved. We confirmed this hypothesis by sequencing two-long terminal repeat (LTR) circle junctions of unintegrated DNA. Based on an infectious foamy virus molecular clone, a set of constructs harboring mutations at the 5' end of the U3 region in the 3' LTR was analyzed for particle export, reverse transcription, and replication. Following transient transfection some mutants were severely impaired in generating infectious virus, while others replicated almost like the wild type. The replication competence of the mutants was unrelated to the cleavability of the newly created U3 end. This became obvious with two mutants both belonging to the high-titer type. One mutant containing a dinucleotide artificially transferred from the right to the left end was trimmed upon integration, while another one with an unrelated dinucleotide in that place was not. The latter construct in particular showed that the canonical TG motif at the beginning of the provirus is not essential for foamy virus integration.

Animals↗

PubMed2003

Replication-competent hybrids between murine leukemia virus and foamy virus.

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.

Amino Acid Sequence↗

PubMed2003

Feline foamy virus genome and replication strategy.

Crucial aspects of the foamy virus (FV) replication strategy have so far only been investigated for the prototypic FV (PFV) isolate, which is supposed to be derived from nonhuman primates. To study whether the unusual features of this replication pathway also apply to more-distantly related FVs, we constructed feline FV (FFV) infectious molecular clones and vectors. It is shown by quantitative RNA and DNA PCR analysis that FFV virions contain more RNA than DNA. Full-length linear DNA was found in extracellular FFV by Southern blot analysis. Similar to PFV, azidothymidine inhibition experiments and the transfection of nucleic acids extracted from extracellular FFV indicated that DNA is the functional relevant FFV genome. Unlike PFV, no evidence was found indicating that FFV recycles its DNA into the nucleus.

Animals↗

PubMed2003

Retrotransposition and cell-to-cell transfer of foamy viruses.

A remarkable feature of the prototype foamy virus (PFV) replication pathway has been reported to consist of the ability to retrotranspose intracellularly with high efficiency (M. Heinkelein, T. Pietschmann, G. Jármy, M. Dressler, H. Imrich, J. Thurow, D. Lindemann, M. Bock, A. Moebes, J. Roy, O. Herchenröder, and A. Rethwilm, EMBO J. 19:3436-3345, 2000). PFV intracellular retrotransposition (IRT) was reported to be enhanced by coexpression of fusion-defective envelope protein. To investigate the possibility of cell-to-cell transfer of PFV genomes, which could mimic IRT, we performed cocultivation experiments with cells transfected with an IRT-competent and marker gene-expressing PFV vector together with cells expressing a different marker and measured cells positive for both markers. The findings corroborated the initial report on IRT of Env-deficient PFV. Furthermore, they indicated that viral cores that have incorporated fusion-deficient Env can be transferred from cell to cell in a cell type-specific manor. One possible explanation consists of a minor alternative cleavage site in Env that can be used to expose the fusion peptide of the Env transmembrane protein, which appears to be required for virus uptake.

Cell Fusion↗