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Retroarray - a Comprehensive Diagnostic DNA Chip for Rapid Detection and Identification of Retroviruses

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Retroarray - a Comprehensive Diagnostic DNA Chip for Rapid Detection and Identification of Retroviruses RetroArray - a comprehensive diagnostic DNA chip for rapid detection and identification of retroviruses Wolfgang Seifarth1 and Christine Leib-Mösch1,2 1 Medical Clinic III, Medical Faculty Mannheim of the University Heidelberg, Mannheim, Germany 2 Helmholtz Center Munich, German Research Center for Environmental Health, Institute of Molecular Virology, Neuherberg, Germany We offer a fast and reliable DNA chip-based assay (RetroArray) for parallel detection and identification of a wide variety of human and mammalian exogenous and endogenous retroviruses (ERV) in biological samples. The assay combines multiplex polymerase chain reaction (PCR) using fluorochrome-modified primers and non-radioactive DNA chip hybridization (figure 1) and features considerable advantages over conventional methods. Advantages at a glance - detects all relevant human, simian and murine retroviruses in a single assay - targets biologically active retroviruses (encoding reverse transcriptase) - user-defined upgrade for any other vertebrate - highest species specificity - high sensitivity (detects as few as ~25 copies/sample) - easy operation and fast performance (results within 20 h) - high throughput capability (automation) - no radioactivity (use of Cy3/Cy5 fluorochromes) TECHNIQUE RetroArray is composed of retrovirus-specific synthetic oligonucleotides as capture probes deposited on glass slides.1,2 Currently, the chip carries 80 spots of human/mammalian endogenous (ERV) and exogenous retrovirus-specific synthetic oligonucleotides. In addition to ERVs common for humans and Old World monkeys (OWM),3,4 RetroArray contains representatives of all macaque-specific endogenous retroviruses identified by the macaque genome sequencing project. Furthermore, an extended mammalian and vertebrate panel with 41 capture probes of rodent and of bovine, ovine, porcine, avian, simian, feline and canine origin is available (see figure 3 for array design).5 As internal controls, a house keeping gene panel (n=5) and several grid localization dots are covalently bound to the glass surface. The array is hybridized with retrovirus-specific DNA probes that are generated by PCR from the sample of interest. Genomic DNA, total RNA or mRNA pools (cDNA) can serve as starting material. PCR is performed using two sets of mixed oligo primers (MOP) labeled with the fluorescent dyes Cy3/Cy5. The highly complex MOP cocktails (patented §) are derived from conserved motifs found in all known reverse transcriptase genes and allow amplification of all retroviral reverse transcriptase-related nucleic acids contained in the sample of interest.1,2 1 RETROARRAY DESIGN AND RESULTS RetroArray has been successfully used for qualitative and quantitative analysis of retroviral activity in human tissue samples (blood, kidney, colon, skin)6 and in comparative studies using paired samples (normal vs. disease).5,7 The assay is useful for identification of retroviral transcripts packaged in retroviral particles released by human cell lines (e.g. T47D human mammary carcinoma cells) and by packaging cell lines of retroviral vector systems.2,8 Thus, for example, RetroArray will be useful to monitor ERV copackaging in therapeutic vector preparations. Furthermore, detection and monitoring of exogenous retroviruses such as HIV-1 and HTLV-1 in infected macrophage cell lines (U373, M8166) was efficient 2 and reliable rendering the assay an excellent tool for testing cell lines for contaminations with exogenous human and animal retroviruses (e.g. squirrel monkey retrovirus, SMRV; murine leukemia virus, MLV; mouse mammary tumor virus, MMTV; foamy viruses). Species specificity was demonstrated by detecting traces of pig endogenous retroviruses (PERV) down to ~25 copies in human DNA and by discriminating between retroviruses originating from different species (mouse/human hybridoma cells, cat, ape, and pig).1,5 2 Tissue-specific activity of human endogenous retroviruses (HERV) RetroArray allows for comprehensive profiling of retroviral expression in cell lines and tissues. Investigation of 19 different human tissues revealed distinct and highly tissue-specific HERV transcription patterns. The characteristic HERV activity profiles can be used to verify the cell type and to monitor cell lines for contaminating cells (e.g. HeLa cells). References 2, 5, 6, 7 3 Comparative analysis of normal and disease-related samples RetroArray allows for detection of disease-related changes in retroviral activities. These may be associated with onset or progression of disease (e.g. breast cancer, neurological diseases). Identified elements may be useful as future molecular markers. References 5, 7, 9 Endogenous retrovirus (ERV) profiling for species identification and detection of contaminating retroviruses RetroArray is an optimal tool for assessing purity and homogeneity of cell lines. Laboratory contaminations with cells of other species or infection with polytropic animal retroviruses will be easily detected due to the excellent species-specificity of the assay. References 5, 7 4 Copackaging of ERV and RCR in retroviral vectors RetroArray is useful to monitor retroviral vector systems for copackaging of replication- competent retroviruses (RCR) or expressed endogenous retroviruses. For example, copackaging of human endogenous retroviruses (HERVs) can be observed in particle preparations from packaging cell lines used for lentivirus- or MLV-based vector systems Reference 8 PERSPECTIVES Over the last couple of years, microarrays (DNA chips) have emerged as powerful and indispensible research tools for detection of microorganisms, for gene expression profiling, mutation and polymorphism analysis in cells and tissues. Employing this technology and a one-step nonradioactive PCR labeling procedure, we set up a high throughput assay for rapid and precise qualitative analysis of retrovirus activity in biological samples. In general, nucleic acids prepared from any biological source, i.e. all types of body fluids, tissues, cells, and cell culture supernatants, can be tested for the presence of retroviral pol (reverse transcriptase) sequences. Our recent publications demonstrate that RetroArray can serve as a powerful tool for various applications in the field of modern biomedicine. Conceivable applications are, for example, survey of xenotransplants before transplantation as well as long-term follow-up of transplant recipients. Thus, RetroArray could contribute to the current efforts to make xenotransplantation safer. Moreover, RetroArray is a valuable tool to detect retroviral transcripts and particles in tissues, cell cultures and cell culture supernatants. Retroviral contaminants in laboratory cell lines, such as squirrel monkey retrovirus (SMRV), or foamy viruses, murine leukemia viruses (MLV), mouse mammary tumor virus (MMTV) will be securely detected and identified. This might be important for the biotechnological production of therapeutic peptides using recombinant DNA technology and human or animal cell lines. Since RetroArray guarantees reliable discrimination between retroviruses originating from different mammalian species it is further suitable for monitoring packaging cell lines and vector preparations with the goal to exclude transfer of copackaged RCR or endogenous retroviruses into target cells. Thus, RetroArray could contribute to safety in gene therapy and protect patients against undesired iatrogeneous transmission of retroviral genetic material. 5 PATENTS §Seifarth, Leib-Mösch, Baust. (1999) Verfahren zum spezifischen Nachweis und zur Identifizierung retroviraler Nukleinsäuren/Retroviren in einem Untersuchungsgut. German (PCT /DE 00/01071) and international (EU, Australia, USA) patents: EP1177320 B1-part AT, EP1177320 B1- part CH/LI, EP1177320 B1- part BE, EP1177320 B1- part DE, EP1177320 B1- part FR, EP1177320 B1- part GB, AU 49104/00 und US10/009,705. Patents cover mixed oligo primer design and usage. REFERENCES 1 Seifarth W., Krause U., Hohenadl C., Baust C., Hehlmann R., and Leib-Mösch C. (2000). Rapid identification of all known reverse transcriptase sequences with a novel versatile detection assay. AIDS Res Hum Retroviruses, 16 (8):721-729. 2 Seifarth W., Spiess B., Zeilfelder U., Speth C., Hehlmann R., and Christine Leib-Mösch C. (2003) Assessment of retroviral activity using a universal retrovirus chip. J Virol Methods 112(1-2):79-91. 3 Stengel A., Roos C., Hunsmann G., Seifarth W., Leib-Mösch C., and Greenwood A. (2006). Expression profiles of endogenous retroviruses in Old World monkeys. J Virology 80(9):4415- 4421. 4 Greenwood A.D., Stengel A., Seifarth W., and Leib-Mösch C. (2005) The distribution of pol containing human endogenous retroviruses in non-human primates, Virology 334(2):203-213. 5 Frank O., Giehl G., Zheng C., Hehlmann R., Leib-Mösch C., and Seifarth W. (2005) Human endogenous retrovirus expression profiles from brains of patients with schizophrenia and bipolar disorders. J Virology, 79:10890-10901. 6 Seifarth W., Frank O., Zeilfelder U., Spiess B., Greenwood A., Hehlmann R., and Leib-Mösch C. (2005) Qualitative analysis of human endogenous retrovirus (HERV) transcriptional activity in human tissues using microarrays. J Virology, 79:341-352. 7 Frank O, Verbeke C, Schwarz N, Fabarius A, Greenwood AD, Hehlmann R, Leib-Mösch C. and Seifarth W. (2008) Variable transcriptional activity of endogenous retroviruses in human breast cancer. J Virology 82(4):1808-1818. 8 Zeilfelder U., Frank O., Sparacio S., Schön U., Bosch V., Seifarth W., and Leib-Mösch C. (2007) The potential of retroviral vectors
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