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Numerous infectious diseases emerged or re-emerged over the past decades. Pathogen identification/detection methods have significantly improved over time; however sample preparation before pathogen detection, development of reliable diagnostic tools as well as proving causality (Hill's criteria) once a pathogen has been identified are still challenging. Next generation sequencing together with PCR amplification techniques are used to hunt for new viruses in acute and chronic diseases with putative infectious etiology.
Improvements of sample preparation for successful detection of viral pathogens from diagnostic samples, development of rapid and reliable diagnostic tools (pathogen detection, immune response) are key elements in our research.
We were recently involved in the identification of a novel human gammaretrovirus, XMRV by using a pan viral DNA microarray developed at the University of California, San Francisco (Urisman, Molinaro, Fischer et al., PLoS Pathog. 2006). XMRV specific sequences were detected in tissue from familial prostate cancer samples harbouring a mutation within the RNASEL gene (R462Q). Sequencing of three different human isolates revealed a high homology to endogenous full length proviral sequences of Mus musculus constituting up to 94% nucleotide identity and allocate this virus to the genus gammaretroviruses, xenotropic Murine Leukemia Virus (xeno-MLV) group. FISH, Immunohistochemistry (IHC) as well as cloning of the integration sites from several patients confirmed that XMRV indeed is the first bonafide human infection with a gammaretrovirus.
Fig. 1: (Urisman A, Molinaro R, Fischer N (co-first authors) et al., 2006, PLoS Pathog.) (A) Fluorescence in situ hybridization using XMRV as a probe in prostate cancer tissue: infected cells are shown in green (SyBr-Green), epithelial cells are stained with a monoclonal antibody against cytokeratin. (B) and (D) Immunhistochemistry using anti-MLV p30 antiserum on prostate cancer tissue.
Subsequent studies of several research groups including ours confirmed the xenotropic host range of XMRV and the necessity of the xenotropic and polytropic receptor protein Xpr1 for virus entry, retroviral restriction by human Apobec 3G and increased LTR-activity in cells derived from the prostate compartment.
Fig. 2: left side: XMRV LTR activity in established human cell lines (293T, HeLa and prostate cancer cell line LNCaP) and primary fibroblasts isolated from prostate tissue (PrSc). Right side: relative mRNA expression levels of human Apobec 3G in human blood cell lines (Jijoye, Raji, Reh and U937), prostate cancer cell lines (LNCaP and DU145), PBMCs from different patients (1, 2) and PrSc cells (a, b). mRNA expression was determined by real-time PCR normalized against three house keeping genes
Our lab focuses on the questions whether XMRV is a putative human pathogen and what are the molecular mechanisms inducing or contributing to pathogenesis. For this purpose XMRV infection is monitored in small animal models.
In 2009, XMRV was identified in up to 68% of PBMC (peripheral blood mononuclear cells) samples from patients with chronic fatigue syndrome while only 3-4% of the control cohort showed signs of XMRV infection (Lombardi et al., Science 2009). PCR data were strengthened by cell dependent as well as cell free transmission of the virus from blood samples of CFS patients to indicator cells. However, several subsequent studies by other labs failed to confirm the PCR data and no virus transmission experiments have been reproduced to date.
Discussed explanations for the observed differences in XMRV prevalence are strain variations of the virus, heterogeneous patient cohorts, non standardized diagnostic assays as well as geographical restriction of the virus. A XMRV blood working group (including labs from the NIH, FDA and CDC) currently investigates these hypotheses.
In December 2010, three publications addressed the risk of contaminations of DNA by traces of mouse DNA (paraffin sections, cell lines or other sources) (Robinson et al., Retrovirology 2010; Oakes et al., Retrovirology 2010) and the risk of false positive PCR products by some commercial amplification kits (Sato et al., Retrovirology 2010). In addition, Hue and colleagues argue that due to the lack of sequence variability of XMRV gene fragments in patients isolates compared to sequence variability identified in a XMRV positive cell line 22Rv1 XMRV might be a laboratory contaminant rather than a true exogenous human virus (Hue et al., Retrovirology et al., 2010)
As PCR contamination is an important criticism (and future studies should include highly sensitive methods to control for contamination), some research data published are not explainable by contamination:
A recent publication suggests vaccines or other biological products produced in mice or mouse cells as a putative mode of XMRV transmission which would explain the high sequence similarity among XMRV genomes, discrepancies in XMRV frequency as well as the geographical restriction described (van der Kuyl et al., Frontiers in Microbiology 2011).
Our lab is interested in epidemiological questions with regard to seroprevalence of XMRV infection, transmission of XMRV and clearance of viral infection. XMRV specific and highly sensitive nucleic acid detection assays for viral detection in non invasive clinical samples will be developed, validated and further improved as a routine diagnostic tool. In addition, serological assays for XMRV detection are currently developed.
Polyomaviruses with SV40 being the best studied virus within this family are small non-enveloped DNA viruses encoding for a large T-Ag which functions as an oncogene. Cells permissive for polyomaviruses support viral replication with high amounts of new viruses being produced and lysis of the host cell. In contrast, in non-permissive cells viral replication is blocked and rarely viral integration into the host genome has been observed resulting in transformed cells.
The genome of Merkel Cell Polyomavirus, MCPyV, a novel human tumor virus identified in 2008 by Pat Moore and Yuan Chang's group (Feng et al., Science 2008), is clonally integrated in tumor tissues of approximately 80% of all Merkel cell carcinoma (MCC) cases, a highly aggressive tumor of the skin which predominantly afflicts elderly and immunosupressed patients.
Fig. 3: Phylogenetic tree of human polyomaviruses, mouse polyomavirus (MuPyV) and monkey SV40 polyomavirus. Human polyomaviruses associated with human diseases are labelled - *-.
All integrated genomes harbor signature mutations in the gene encoding large T-Antigen (LT-Ag), a multifunctional protein which mediates replication of polyomavirus episomes, but which also targets tumor suppressor proteins such as Retinoblastoma protein (Rb) and p53. Sequence analysis demonstrated that large T-Ags from MCCs contain inactivating mutations containing the Rb protein binding site.
We have identified a suitable contingent of MCPyV-infected MCC cell lines and have characterized the LT-Ag mutations of the integrated viral genomes (Fischer, Brandner et al., Int. J. of Cancer 2010).
Fig. 4: Schematic representation of the functional domains of the Large T-Ag.Position of premature STOP codons identified in MCC cell lines (MKL-1; MCCL-11; MCCL-12; MCCL-3) are indicated.
We comprehensively investigate MCPyV T-Ag function in order to elucidate their overall transforming potential, the contribution of individual T-Ag domains to the transformation process, the cell type dependency of T-Ag mediated transformation, the significance of the hallmark large T-Antigen (LT-Ag) truncations observed in MCC tissues, and the contribution of constitutive T-Ag expression to the sustained proliferation of MCC cells. We address these questions in heterologous cell systems as well as MCC-derived cell lines; as such cells represent the best available in vitro model system for MCC.
Being integrated in the interdisciplinary graduate program - Hamburg School for Structure and dynamics in infection (SDI) - funded by the Landesexzellenzinitiative Hamburg, we investigate structural properties of MCPyV LT-Ag by applying structure biology methods (Dynamic Light Scattering, Small-angle X-ray scattering, X-ray crystallography).
| Position | Name | Phone | Fax | |
|---|---|---|---|---|
| Group leader | PD Dr. rer. nat. N. Fischer | 58184 | n.fischer@uke.de | |
| MTA | C. Schmidt | 54663 | cla.schmidt@uke.de | |
| Postdoc | Dr. rer. nat. K. Stieler | 54663 | k.stieler@uke.de | |
| Graduate student | M. Sc. S. Borchert | 54663 | s.borchert@uke.de | |
| Graduate student | M. Leitz | 54663 | ||
| Graduate student | S. Schindler | 54663 |
Stieler K, Fischer N.
Apobec 3G efficiently reduces infectivity of the human exogenous gammaretrovirus XMRV.
PLoS One. 2010 Jul 23;5(7):e11738.
Fischer N, Schulz C, Stieler K, Hohn O, Lange C, Drosten C, Aepfelbacher M.
Xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract.
Emerg Infect Dis. 2010 Jun;16(6):1000-2.
Stieler K, Schulz C, Lavanya M, Aepfelbacher M, Stocking C, Fischer N.
Host range and cellular tropism of the human exogenous gammaretrovirus XMRV.
Virology. 2010 Mar 30;399(1):23-30. Epub 2010 Jan 27.
Fischer N, Brandner J, Fuchs F, Moll I, Grundhoff A.
Detection of Merkel cell polyomavirus (MCPyV) in Merkel cell carcinoma cell lines: Cell morphology and growth phenotype do not reflect presence of the virus.
Int J Cancer. 2009 Sep 8.
Fischer N, Hellwinkel O, Schulz C, Chun FK, Huland H, Aepfelbacher M, Schlomm T.
Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer.
J Clin Virol. 2008 Nov;43(3):277-83. Epub 2008 Sep 27.
Urisman A, Molinaro R J, Fischer N, Plummer S J, Casey G, Klein E A, Malathi K, Magi-Galluzzi C, Tubbs R R, Ganem D, Silverman R H, DeRisi J L.
Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.
PLoS Pathog. 2006 March;2(3):e25
Brune C, Munchel S E, Fischer N, Podtelejnikov A V, Weis K.
Yeast poly(A)-binding protein Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export.
RNA. 2005 April;11(4):517-531.
Fischer N, Weis K.
The DEAD box protein Dhh1 stimulates the decapping enzyme Dcp1.
EMBO J. 2002 June;21(11):2788-2797.
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