Sebastian Voigt

Publications of Sebastian Voigt

• Molecular analysis of varicella vaccines and varicella-zoster virus from vaccine-related skin lesions.

  Authors: Sonja Thiele, Aljona Borschewski, Judit Küchler, Marc Bieberbach, Sebastian Voigt, Bernhard Ehlers

  Clinical and vaccine immunology : CVI. 05/2011; 18(7):1058-66.

  To prevent complications that might follow an infection with varicella-zoster virus (VZV), the live attenuated Oka strain (V-Oka) is administered to children in many developed countries. ThreeTo prevent complications that might follow an infection with varicella-zoster virus (VZV), the live attenuated Oka strain (V-Oka) is administered to children in many developed countries. Three vaccine brands (Varivax from Sanofi Pasteur MSD; Varilrix and Priorix-Tetra, both from Glaxo-Smith-Kline) are licensed in Germany and have been associated with both different degrees of vaccine effectiveness and adverse effects. To identify genetic variants in the vaccines that might contribute to rash-associated syndromes, single nucleotide polymorphism (SNP) profiles of variants from the three vaccines and rash-associated vaccine-type VZV from German vaccinees were quantitatively compared by PCR-based pyrosequencing (PSQ). The Varivax vaccine contained an estimated 3-fold higher diversity of VZV variants, with 20% more wild-type (wt) SNPs than Varilrix and Priorix-Tetra. These minor VZV variants in the vaccines were identified by analyzing cloned full-length open reading frame (ORF) orf62 sequences by chain termination sequencing and PSQ. Some of these sequences amplified from vaccine VZV were very similar or identical to those of the rash-associated vaccine-type VZV from vaccinees and were almost exclusively detected in Varivax. Therefore, minorities of rash-associated VZV variants are present in varicella vaccine formulations, and it can be concluded that the analysis of a core set of four SNPs is required as a minimum for a firm diagnostic differentiation of vaccine-type VZV from wt VZV.

• Switch to high-level virus replication and HLA class I upregulation in differentiating megakaryocytic cells after infection with pathogenic hantavirus.

  Authors: Nina Lütteke, Martin J Raftery, Pritesh Lalwani, Min-Hi Lee, Thomas Giese, Sebastian Voigt, Norbert Bannert, Harald Schulze, Detlev H Krüger, Günther Schönrich

  Virology. 09/2010; 405(1):70-80.

  Hantaan virus (HTNV), the prototype member of the Hantavirus genus in the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS) in humans. Hemorrhage is due to endothelial barrierHantaan virus (HTNV), the prototype member of the Hantavirus genus in the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS) in humans. Hemorrhage is due to endothelial barrier damage and a sharp decrease in platelet counts. The mechanisms underlying HTNV-associated acute thrombocytopenia have not been elucidated so far. Platelets are produced by mature megakaryocytes that develop during megakaryopoiesis. In this study, we show that HTNV targets megakaryocytic cells whereas rather non-pathogenic hantaviruses did not infect this cell type. After induction of differentiation megakaryocytic cells switched from low-level to high-level HTNV production without reduction in cell survival or alteration in differentiation. However, increased HTNV replication resulted in strong upregulation of HLA class I molecules although HTNV escaped type I interferon (IFN)-associated innate responses. Taken together, HTNV efficiently replicates in differentiating megakaryocytic cells resulting in upregulation of HLA class I molecules, the target structures for cytotoxic T cells (CTLs).

• Improved detection of mutated human cytomegalovirus UL97 by pyrosequencing.

  Authors: Birgit Schindele, Luise Apelt, Jörg Hofmann, Andreas Nitsche, Detlef Michel, Sebastian Voigt, Thomas Mertens, Bernhard Ehlers

  Antimicrobial agents and chemotherapy. 09/2010; 54(12):5234-41.

  Ganciclovir (GCV) resistance frequently occurs upon prolonged treatment of ongoing active human cytomegalovirus (HCMV) infection in individuals with immature or compromised immune functions (e.g.,Ganciclovir (GCV) resistance frequently occurs upon prolonged treatment of ongoing active human cytomegalovirus (HCMV) infection in individuals with immature or compromised immune functions (e.g., recipients of solid-organ and hematopoietic stem cell transplants). Using pyrosequencing (PSQ), we established fast and sensitive detection of GCV resistance-associated mutations occurring in the HCMV open reading frame UL97. These mutations have been repeatedly associated with clinical treatment failure. We designed four PSQ assays and evaluated them by analyzing mixtures of plasmids or bacterial artificial chromosome-derived viruses containing UL97 wild-type and mutant sequences. A minimum level of 6% mutant sequence variants could be detected in these mixtures. In order to further evaluate the novel PSQ assays, we tested clinical specimens from patients with active HCMV infections. The results were compared with those obtained by conventional dideoxy chain terminator sequencing. As the PSQ method was more sensitive in detecting minor HCMV mutant fractions in a wild-type population, it is suggested that pyrosequencing is a useful tool for the early detection of emerging GCV-resistant HCMV in GCV-treated patients.

• Identification of a novel betaherpesvirus in Mus musculus.

  Authors: Alla Teterina, Dania Richter, Franz-Rainer Matuschka, Bernhard Ehlers, Sebastian Voigt

  Virology journal. 12/2009; 6(1):225.

  ABSTRACT: Rodent betaherpesviruses vary considerably in genomic content, and these variations can result in a distinct pathogenicity. Therefore, the identification of unknown betaherpesviruses inABSTRACT: Rodent betaherpesviruses vary considerably in genomic content, and these variations can result in a distinct pathogenicity. Therefore, the identification of unknown betaherpesviruses in house mice (Mus musculus), the most important rodent host species in basic research, is of importance. During a search for novel herpesviruses in house mice using herpesvirus consensus PCR and attempts to isolate viruses in tissue culture, we identified a previously unknown betaherpesvirus. The primary PCR search in mouse organs revealed the presence of known strains of murine cytomegalovirus (Murid herpesvirus 1) and of Mus musculus rhadinovirus 1 only. However, the novel virus was detected after incubation of organ pieces in fibroblast tissue culture and subsequent PCR analysis of the supernatants. Long distance PCR amplification including the DNA polymerase and glycoprotein B genes revealed a 3.4 kb sequence that was similar to sequences of rodent cytomegaloviruses. Pairwise sequence comparisons and phylogenetic analyses showed that this newly identified murine virus is most similar to the English isolate of rat cytomegalovirus, thereby raising the possibility that two distinct CMV lineages have evolved in both Mus musculus and Rattus norvegicus.

• High genotypic diversity and a novel variant of human cytomegalovirus revealed by combined UL33 / UL55 genotyping with broad-range PCR.

  Authors: Merlin Deckers, Jorg Hofmann, Karl-Anton Kreuzer, Henrike Reinhard, Abigail Edubio, Hartmut Hengel, Sebastian Voigt, Bernhard Ehlers

  Virology journal. 11/2009; 6(1):210.

  ABSTRACT: The known strains of human cytomegalovirus (HCMV) represent genotypic variants of a single species, and HCMV genotypic variability has been studied in order to reveal correlations betweenABSTRACT: The known strains of human cytomegalovirus (HCMV) represent genotypic variants of a single species, and HCMV genotypic variability has been studied in order to reveal correlations between different disease patterns and the presence of certain HCMV genotypes, either as single or as multiple infections. The methods used for the detection of HCMV genotypes have not always been sophisticated enough to achieve complete comprehensiveness, mainly because only one genotype is usually detected in a certain specimen, due to primer specificity and genome copy number. To improve detection of variant HCMV genotypes in mixed infections, we developed PCR assays with degenerate primers targeting two variable HCMV genes, glycoprotein B (gB, UL55) and the G-protein-coupled receptor gene UL33. Primers were designed to bind conserved sites in the genomes of HCMV variants and great ape CMVs. To analyse if samples contained one or more HCMV genotypic variants, PCR assays were supplemented with oligonucleotides containing locked nucleic acids. This broad-range PCR methodology and subsequent sequence analysis detected all gB/UL55 and UL33 genotypic variants known to date in primary clinical specimens, but also revealed that many samples contained genotype mixtures. Importantly, a novel UL33 genotypic variant could be discovered in several specimens, and one HCMV isolate was plaque-purified containing the novel UL33 genotype and a so far undescribed variant of gB.

• Deletion of the rat cytomegalovirus immediate early 1 gene results in a virus capable of establishing latency but with lower levels of acute virus replication and latency that compromise reactivation efficiency.

  Authors: Gordon R. Sandford, Uwe Schumacher, Jakob Ettinger, Wolfram Brune, Gary S Hayward, William H Burns, Sebastian Voigt

  The Journal of general virology. 11/2009;

  The IE1 and IE2 proteins encoded by the major immediate-early (MIE) transcription unit of cytomegaloviruses are thought to play key roles in the switch between latent and lytic cycle infection.The IE1 and IE2 proteins encoded by the major immediate-early (MIE) transcription unit of cytomegaloviruses are thought to play key roles in the switch between latent and lytic cycle infection. Whilst IE2 is essential for triggering the lytic cycle, the exact roles of IE1 have not been resolved. An MIE-exon 4 deleted rat cytomegalovirus (DeltaIE1) failed to synthesize the IE1 protein and did not disperse promyelocytic leukemia bodies (PML) early post-infection, but it was still capable of normal replication in fibroblast cell culture. However, DeltaIE1 had diminished ability to infect salivary glands persistently in vivo and to reactivate from spleen explant cultures ex vivo. Quantitation of viral genomes in spleens of infected animals revealed a reduced amount of DeltaIE1 virus produced during acute infection, suggesting a role for IE1 as a regulator in establishing a chronic or persistent infection, rather than in more directly influencing the latency or reactivation processes.

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• Cell cycle independent expression of immediate early gene 3 results in a G1 and G2 arrest in MCMV-infected cells.

  Authors: Lüder Wiebusch, Anke Neuwirth, Linus Grabenhenrich, Sebastian Voigt, Christian Hagemeier

  Journal of virology. 08/2008;

  The infectious cycle of human cytomegalovirus is intricately linked to the host's cell cycle. Viral gene expression can be initiated only in G0/G1 phase. Once expressed, the immediate early geneThe infectious cycle of human cytomegalovirus is intricately linked to the host's cell cycle. Viral gene expression can be initiated only in G0/G1 phase. Once expressed, the immediate early gene product IE2 prevents cellular DNA synthesis arresting infected cells with a G1 DNA content. This function is required for efficient viral replication in vitro. A prerequisite for addressing its in vivo relevance is the characterization of cell cycle regulatory activities of CMV species for which animal models have been established. Here, we show that murine CMV (MCMV), like HCMV, has a strong anti-proliferative capacity and arrests cells in G1. Unexpectedly, and in contrast to HCMV, MCMV can also block cells that have passed through S phase by arresting them in G2. Moreover, MCMV can also replicate in G2 cells. This is made possible by the cell cycle independent expression of MCMV immediate early genes. Transfection experiments show that of several MCMV candidate genes only immediate early gene 3 (ie3), the homologue of HCMV-IE2, exhibits cell cycle arrest activity. Accordingly, a MCMV ie3 deletion mutant has lost the ability to arrest cells either in G1 or G2. Thus, despite interspecies variations in the cell cycle dependence of viral gene expression the central theme of HCMV IE2-induced cell cycle arrest is conserved in the murine counterpart raising the possibility to study its physiological relevance at the level of the whole organism.

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• Murine cytomegalovirus m38.5 protein inhibits Bax-mediated cell death.

  Authors: Igor Jurak, Uwe Schumacher, Hrvoje Simic, Sebastian Voigt, Wolfram Brune

  Journal of virology. 06/2008; 82(10):4812-22.

  Many viruses encode proteins that inhibit the induction of programmed cell death at the mitochondrial checkpoint. Murine cytomegalovirus (MCMV) encodes the m38.5 protein, which localizes toMany viruses encode proteins that inhibit the induction of programmed cell death at the mitochondrial checkpoint. Murine cytomegalovirus (MCMV) encodes the m38.5 protein, which localizes to mitochondria and protects human HeLa cells and fibroblasts from apoptosis triggered by proteasome inhibitors but not from Fas-induced apoptosis. However, the ability of this protein to suppress the apoptosis of murine cells and its role during MCMV infection have not been investigated previously. Here we show that m38.5 is expressed at early time points during MCMV infection. Cells infected with MCMVs lacking m38.5 showed increased sensitivity to cell death induced by staurosporine, MG132, or the viral infection itself compared to the sensitivity of cells infected with wild-type MCMV. This defect was eliminated when an m38.5 or Bcl-X(L) gene was inserted into the genome of a deletion mutant. Using fibroblasts deficient in the proapoptotic Bcl-2 family proteins Bak and/or Bax, we further demonstrated that m38.5 protected from Bax- but not Bak-mediated apoptosis and interacted with Bax in infected cells. These results consolidate the role of m38.5 as a viral mitochondrion-localized inhibitor of apoptosis and its functional similarity to the human cytomegalovirus UL37x1 gene product. Although the m38.5 gene is not homologous to the UL37x1 gene at the sequence level, m38.5 is conserved among rodent cytomegaloviruses. Moreover, the fact that MCMV-infected cells are protected from both Bak- and Bax-mediated cell death suggests that MCMV possesses an additional, as-yet-unidentified mechanism to block Bak-mediated apoptosis.

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• Identification of novel rodent herpesviruses, including the first gammaherpesvirus of Mus musculus.

  Authors: Bernhard Ehlers, Judit Küchler, Nezlisah Yasmum, Güzin Dural, Sebastian Voigt, Jonas Schmidt-Chanasit, Thomas Jäkel, Franz-Rainer Matuschka, Dania Richter, Sandra Essbauer, David J Hughes, Candice Summers, Malcolm Bennett, James P Stewart, Rainer G Ulrich

  Journal of virology. 09/2007; 81(15):8091-100.

  Rodent herpesviruses such as murine cytomegalovirus (host, Mus musculus), rat cytomegalovirus (host, Rattus norvegicus), and murine gammaherpesvirus 68 (hosts, Apodemus species) are important toolsRodent herpesviruses such as murine cytomegalovirus (host, Mus musculus), rat cytomegalovirus (host, Rattus norvegicus), and murine gammaherpesvirus 68 (hosts, Apodemus species) are important tools for the experimental study of human herpesvirus diseases. However, alphaherpesviruses, roseoloviruses, and lymphocryptoviruses, as well as rhadinoviruses, that naturally infect Mus musculus (house mouse) and other Old World mice are unknown. To identify hitherto-unknown rodent-associated herpesviruses, we captured M. musculus, R. norvegicus, and 14 other rodent species in several locations in Germany, the United Kingdom, and Thailand. Samples of trigeminal ganglia, dorsal root ganglia, brains, spleens, and other organs, as well as blood, were analyzed with a degenerate panherpesvirus PCR targeting the DNA polymerase (DPOL) gene. Herpesvirus-positive samples were subjected to a second degenerate PCR targeting the glycoprotein B (gB) gene. The sequences located between the partial DPOL and gB sequences were amplified by long-distance PCR and sequenced, resulting in a contiguous sequence of approximately 3.5 kbp. By DPOL PCR, we detected 17 novel betaherpesviruses and 21 novel gammaherpesviruses but no alphaherpesvirus. Of these 38 novel herpesviruses, 14 were successfully analyzed by the complete bigenic approach. Most importantly, the first gammaherpesvirus of Mus musculus was discovered (Mus musculus rhadinovirus 1 [MmusRHV1]). This virus is a member of a novel group of rodent gammaherpesviruses, which is clearly distinct from murine herpesvirus 68-like rodent gammaherpesviruses. Multigenic phylogenetic analysis, using an 8-kbp locus, revealed that MmusRHV1 diverged from the other gammaherpesviruses soon after the evolutionary separation of Epstein-Barr virus-like lymphocryptoviruses from human herpesvirus 8-like rhadinoviruses and alcelaphine herpesvirus 1-like macaviruses.

• Cytomegalovirus evasion of innate immunity by subversion of the NKR-P1B:Clr-b missing-self axis.

  Authors: Sebastian Voigt, Aruz Mesci, Jakob Ettinger, Jason H Fine, Peter Chen, Wayne Chou, James R Carlyle

  Immunity. 06/2007; 26(5):617-27.

  Cytomegaloviruses are known to encode several gene products that function to subvert MHC-dependent immune recognition. Here we characterize a rat cytomegalovirus (RCMV) C-type lectin-like (RCTL) geneCytomegaloviruses are known to encode several gene products that function to subvert MHC-dependent immune recognition. Here we characterize a rat cytomegalovirus (RCMV) C-type lectin-like (RCTL) gene product with homology to the Clr ligands for the NKR-P1 receptors. RCMV infection rapidly extinguished host Clr-b expression, thereby sensitizing infected cells to killing by natural killer (NK) cells. However, the RCTL protein functioned as a decoy ligand to protect infected cells from NK killing via direct interaction with the NKR-P1B inhibitory receptor. In vivo, an RCTL mutant virus displayed diminished virulence in an NK-dependent and strain-specific manner, suggesting that host NKR-P1 polymorphisms have evolved to avert the viral decoy mechanism while maintaining Clr-b recognition to preserve self tolerance. These findings reveal a unique strategy adopted by cytomegaloviruses to evade MHC-independent self-nonself discrimination. The existence of lectin-like genes in several poxviruses suggests that this may represent a common theme for viral evasion of innate immunity.

• Fatal reactivation of postnatal cytomegalovirus infection with rapid emergence of ganciclovir resistance in an infant after allogeneic stem cell transplantation.

  Authors: Sebastian Voigt, Detlef Michel, Olivia Kershaw, Jörn-Sven Kühl, Thomas Mertens, Wolfram Ebell, Helga Meisel

  Journal of clinical microbiology. 08/2005; 43(7):3551-4.

  Human cytomegalovirus (HCMV) can cause serious problems after hematopoietic stem cell transplantation. The death of a pediatric transplant recipient after reactivation of a postnatal HCMV infectionHuman cytomegalovirus (HCMV) can cause serious problems after hematopoietic stem cell transplantation. The death of a pediatric transplant recipient after reactivation of a postnatal HCMV infection with bilateral retinitis and pneumonitis is described. Sequencing of the HCMV UL97 region revealed a compartment-specific mutation (H520Q) in urine conferring ganciclovir resistance.

• The English strain of rat cytomegalovirus (CMV) contains a novel captured CD200 (vOX2) gene and a spliced CC chemokine upstream from the major immediate-early region: further evidence for a separate evolutionary lineage from that of rat CMV Maastricht.

  Authors: Sebastian Voigt, Gordon R. Sandford, Gary S Hayward, William H Burns

  The Journal of general virology. 03/2005; 86(Pt 2):263-74.

  Sequence data for eight genes, together with time-course Northern blotting and 3'- and 5'-RACE (rapid amplification of cDNA ends) analysis for some mRNAs from a 12 kb region upstream from the majorSequence data for eight genes, together with time-course Northern blotting and 3'- and 5'-RACE (rapid amplification of cDNA ends) analysis for some mRNAs from a 12 kb region upstream from the major immediate-early (MIE) genes of the English isolate of rat cytomegalovirus (RCMV), are presented. The results identified important differences compared to both murine cytomegalovirus (MCMV) and the Maastricht isolate of RCMV. A striking finding is the presence of a highly conserved, rightwards-oriented homologue of the rat cellular CD200 (OX2) gene immediately to the right of the MIE region, which replaces either the leftwards-oriented AAV REP gene of RCMV (Maastricht) or the upstream spliced portions of the immediate-early 2 gene (ie2) in MCMV. From the presence of other homologues of MCMV- and RCMV-specific genes, such as the beta-chemokine MCK-2, SGG1 and an Fcgamma receptor gene, as reported here, the basic architecture of the MIE region (reported previously) and the level of IE2 and DNA polymerase (POL) protein conservation in phylogenetic analyses, it is clear that the English strain of RCMV is also a member of the genus Muromegalovirus, but is a beta-herpesvirus species that is very distinct from both MCMV and RCMV (Maastricht). Both the lack of a CD200 homologue in the other two rodent viruses and the depth of sequence divergence of the rodent CMV IE2 and POL proteins suggest that these three viruses have evolved as separate species in the genus Muromegalovirus since very early in the host rodent lineage.

• High genotypic diversity and a novel variant of human cytomegalovirus revealed by combined UL33/UL55 genotyping with broad-range PCR

  Authors: Merlin Deckers, Jörg Hofmann, Karl-Anton Kreuzer, Henrike Reinhard, Abigail Edubio, Hartmut Hengel, Sebastian Voigt, Bernhard Ehlers

  The known strains of human cytomegalovirus (HCMV) represent genotypic variants of a single species, and HCMV genotypic variability has been studied in order to reveal correlations between differentThe known strains of human cytomegalovirus (HCMV) represent genotypic variants of a single species, and HCMV genotypic variability has been studied in order to reveal correlations between different disease patterns and the presence of certain HCMV genotypes, either as single or as multiple infections. The methods used for the detection of HCMV genotypes have not always been sophisticated enough to achieve complete comprehensiveness, mainly because only one genotype is usually detected in a certain specimen, due to primer specificity and genome copy number. To improve detection of variant HCMV genotypes in mixed infections, we developed PCR assays with degenerate primers targeting two variable HCMV genes, glycoprotein B (gB, UL55) and the G-protein-coupled receptor gene UL33. Primers were designed to bind conserved sites in the genomes of HCMV variants and great ape CMVs. To analyse if samples contained one or more HCMV genotypic variants, PCR assays were supplemented with oligonucleotides containing locked nucleic acids. This broad-range PCR methodology and subsequent sequence analysis detected all gB/UL55 and UL33 genotypic variants known to date in primary clinical specimens, but also revealed that many samples contained genotype mixtures. Importantly, a novel UL33 genotypic variant could be discovered in several specimens, and one HCMV isolate was plaque-purified containing the novel UL33 genotype and a so far undescribed variant of gB.