IACFS/ME Biennial International Conference Ottawa, Ontario, Canada Abstracts from General Session September 23, 2011 Gammaretroviruses of Mice and Their Links to Prostate Cancer and CFS/ME Christine Kozak, Ph.D. Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Bethesda, MD, 20892- 0460, USA Gammaretroviruses of three distinct host range tropisms have been isolated from the laboratory mouse. These viruses differ in receptor usage, distribution among wild mouse species and strains of laboratory mice, pathogenicity and sensitivity to host restriction factors. Two of these three host range groups, the xenotropic and polytropic mouse leukemia viruses (together termed XP-MLVs) are widely distributed in house mouse species, mice that live in closest contact with humans. XP-MLVs rely on the XPR1 receptor for entry into cells as does the xenotropic murine leukemia virus-related virus (XMRV) initially identified in human patient samples. Despite their initial description as viruses incapable of infecting mouse cells, the xenotropic viruses have the broadest host range of the MLVs. Nearly all nonrodent mammals are susceptible to X-MLVs, as are all wild mouse species and some inbred strains of laboratory mice. Their XPR1 receptor is highly polymorphic, and there are 5 functional variants of Xpr1 in Mus species and laboratory mouse strains that differ in their ability to support entry of XMRV and various isolates of XP-MLVs. The distribution of XP-MLVs and Xpr1 variants in wild mouse populations provides a good example of how diversifying selection can be driven by genetic conflicts. Restrictive receptor variants evolved in Eurasian house mouse populations exposed to XP-MLV infection suggesting that positive selection favors antiviral alleles in virus-infected species. The ecotropic and polytropic MLVs have long been linked to disease induction in mice, and the discovery that all wild mice and some laboratory strains are also susceptible to X-MLV has made it possible to examine the disease inducing potential of these viruses in mice as well as in other model systems. X-MLVs are capable of establishing infection in mice carrying permissive XPR1 alleles, but X-MLV does not induce or accelerate disease in mice with permissive receptors inoculated as adults or neonates, and X-MLVs do not readily establish productive infection in monkeys. Host factors that restrict retroviruses effectively limit virus spread and disease induction in mice and other species. Session: VIROLOGY RESEARCH Session Chair: Jose Montoya, M.D. Multi-laboratory Evaluations of XMRV Detection Assays Graham Simmons, Ph.D. John M. Coffin2, Indira K. Hewlett3, Shyh-Ching Lo4, Judy A. Mikovits5, William H. Switzer6, Jeffrey M. Linnen7, Francis Ruscetti8, Simone A. Glynn9 and Michael P. Busch1 1Blood Systems Research Institute and Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94118, USA 2National Cancer Institute and Department of Molecular Biology & Microbiology and Program in Genetics, Tufts University, Boston, MA 02111, USA. 3Office of Blood Research and Review, FDA, Bethesda, MD 20892, USA 4Division of Cellular and Gene Therapies and Division of Human Tissues, FDA, Bethesda, MD 20892, USA 5Whittemore Peterson Institute and University of Nevada, Reno, NV 89557, USA Bulletin of the IACFS/ME 112 6Division of HIV/AIDS Prevention, CDC, Atlanta, GA 30333, USA 7Gen-Probe Incorporated, San Diego, CA, USA 8Laboratory of Experimental Immunology, National Cancer Institute- Frederick, Frederick, MD 21701, USA 9Transfusion Medicine and Cellular Therapeutics Branch, NHLBI, Bethesda, MD 20892, USA Background: The Blood XMRV Scientific Research Working Group was established to design and coordinate collaborative studies to investigate the prevalence of XMRV in blood donors using standardized XMRV assays. Materials And Methods: A multi-phase study has been designed to evaluate XMRV nucleic acid and serological detection assays in terms of sensitivity, specificity and reproducibility; assess assay performance on various specimen types represented in existing blood donor/recipient repositories, and determine the prevalence of XMRV in blood donors. Phase I involved production of whole blood (WB) and plasma analytical performance panels spiked with XMRV infected cells or virus, respectively. These panels were tested in a blinded fashion using XMRV nucleic acid amplification testing (NAT) developed by seven participating laboratories. Phase II represented pilot studies to compare XMRV detection using frozen PBMCs, WB and plasma derived from individuals identified as XMRV viremic in a previous study. Additionally, serology was performed on plasma by two laboratories. Phase III involves further evaluation of the clinical sensitivity and specificity of candidate NAT, serology and culture assays by using a blinded panel of 15 pedigreed positive samples, together with pedigreed negative samples and spiked positive controls. Results: In phase I, all laboratories detected at least 136 proviral copies/ml and 5/7 assays demonstrated even more sensitive limits of detection. 5/7 plasma RNA assays performed similarly, with limits of detection of 80 RNA copies/ml or less. The initial unblinded pilot study in phase II resulted in two laboratories detecting MLV-like sequences in the plasma, but not PBMCs or WB, from all four subjects. A third laboratory detected no viral sequences. A second, blinded, pilot study using the same four subjects and two validated negative controls was less conclusive, with three laboratories detecting no viral sequences with any of the samples. A FACS-based serological assay detected antibodies in 3/4 XMRV-positive individuals, but also in 1/2 negative controls. A western-based assay found no evidence of serology in any sample. Results from Phase III are expected soon. Conclusions: The Blood XMRV SRWG has established a collaboration between many of the laboratories conducting research into XMRV and its detection in blood and has initiated steps to compare performance of XMRV assays using analytical and clinical panels comprised of blood samples from XMRV-positive and negative pedigreed subjects. Detection Of Anti-XMRV Antibodies In Serum of CFS Patients and Healthy Blood Donors in Belgium Kenny De Meirleir, M.D. Marc Frémont2, Svetlana Khaliboulina3, Vincent C. Lombardi3, Cassandra Puccinelli3, Kristine Metzger2, Judy A. Mikovits3 1. Department of Human Physiology, Vrije Universiteit Brussel, Brussels, Belgium 2. RED Laboratories, Zellik, Belgium 3. Whittemore Peterson Institute, Reno, Nevada, USA Objectives: Xenotropic murine leukemia virus–related virus (XMRV) is a new human gammaretrovirus originally identified in prostate cancer patients with a deficiency in the antiviral enzyme RNase L. An association has been made between XMRV and Chronic Fatigue Syndrome (CFS), with a 2009 study reporting the presence of XMRV DNA in the blood of 67% of CFS patients, whereas only 3,7% of healthy controls tested positive. In 2010 another study detected murine leukemia virus (MLV)-like GAG sequences in 86,5% of CFS patients, versus only 6,8% of healthy blood donors. A number of other studies, however, have failed to detect XMRV DNA in the blood of CFS patients. The objectives of this study were to investigate the association between CFS and XMRV in a Belgian population of patients, and to estimate the prevalence of XMRV infections in the general population in Belgium. Methods: A flow cytometry-based assay was used to detect the presence of circulating anti-XMRV antibodies in the serum of 84 Belgian CFS patients. A subgroup of these patients (21) have developed CFS after receiving a blood transfusion. Serum obtained from 44 Red Cross healthy blood donors was also tested. Samples were collected in Belgium and sent, blinded, to the Whittemore Peterson Institute in Reno for analysis. Bulletin of the IACFS/ME 113 Results: 48 out of 84 patients (57%) presented circulating antibodies against XMRV (10 out of the 21 patients who received a transfusion). In contrast, only 7 out of 44 controls had anti-XMRV antibodies (16%). Conclusions: The higher prevalence of serology positives in the patient population, compared to the controls, supports the idea that XMRV is involved in the pathogenesis of CFS. The finding that 16% of healthy blood donors present evidence of infection with XMRV or a related virus raises questions regarding the need to screen blood donors for asymptomatic XMRV infections. Prof. Kenny De Meirleir, M.D., Ph.D., Department of Human Physiology, Vrije Universiteit Brussel, Pleinlaan 2, B- 1051 Brussels Belgium, Email: [email protected] Detection of MLV-like gag Sequences in Blood and Cell Lines Incubated With Plasma From CFS Patients and Controls Maureen Hanson, Ph.D. L.L. Lee1, L. Lin1, D.E. Bell2, D. Ruppert3, S. Levine4, D.S. Bell5. 1Cornell University, Molecular Biology and Genetics, Ithaca NY, 2State University of New York, Dept. of Medical Anthropology, Buffalo NY, 3Cornell University, School of Operations Research and Information Engineering, Ithaca NY, 4Private Practice, New York City, 5State University of New York, Dept. of Pediatrics, Buffalo NY Objectives: To determine whether viruses related to XMRV could be detected in peripheral blood from adult subjects who are either ill with CFS, are recovered from CFS, or have no history of a CFS diagnosis. Methods: Subjects were divided into five groups. Ten subjects were severely ill with CFS, ten met Fukuda criteria at one time but