Friday, March 25, 2011

Dr Nicole Fischer: Some published research data are not explainable by XMRV contamination

Dr Nicole Fischer, Universitätsklinikum Hamburg-Eppendorf:

Molecular characterization of a novel gammaretrovirus (XMRV): XMRV pathogenesis studies

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.

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.

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.

Current controversy about XMRV

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:

Identification of human integration sites described by Dong and colleagues (Dong et al., PNAS 2007) can not be amplified from 22Rv1 cells (our own observation).
FISH detection of XMRV and XMRV protein expression in serial sections of patient material (Urisman et al., PLoS Pathog. 2006).

A recent publication suggests vaccines or other biological products produced in mice or mouse cells as a putative mode of XMRV transmissionwhich 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.

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