Plasma samples randomly collected from healthy donors (n = 500) at the Japanese Red Cross Osaka Blood Center between December 2006 and May 2009 were subjected to XMRV Ab screening. All donors had negative results in the routine tests at the Center: antigen testing of hepatitis B virus (HBV) and human parvovirus B19; Ab testing against HBV, hepatitis C virus (HCV), HIV-1, HIV-2, HTLV-1, and syphilis; nucleic acids of HIV-1, HIV-2, HBV, and HCV. All procedures in the donor screening study were performed according to the guidelines of the Japanese Red Cross Society, which do not permit the detection of nucleic acids from unapproved viruses.
All patients with PC enrolled in this study (n = 67) received medical treatment at Nishiwaki City Hospital (Hyogo Prefecture, Japan) between December 2007 and December 2009, when plasma samples were collected, and provided written informed consent. Whole blood samples in ethylenediaminetetraacetic acid (EDTA) were separated by centrifugation, and the plasma was stored at -80°C until use. PBMCs of the patients who tested positive for XMRV Abs and RNA were used for RNASEL sequencing and viral isolation. This study was approved by the ethical committee of Nishiwaki City Hospital.
CFS patients in this study fulfilled the 1994 CDC Fukuda criteria  and received medical treatment at the Fatigue Clinic Center, Osaka City University Graduate School of Medicine, Osaka, Japan between April and August 2010. Most of the patients were female (69%) with an age distribution of 17-62 years (mean, 38 years). The mean interval from disease onset to blood collection was 126.5 months (11-337 months). Duplicated tubes of 4 ml of whole blood in EDTA were used for Ab screening and genomic PCR assay. Whole blood samples were also collected into sodium heparin tubes (Becton Dickinson, Franklin Lakes, NJ) for cell culture. All blood samples were conveyed to the Japanese Red Cross Osaka Blood Center and genomic DNA was purified from them on the same day. Three aliquots of genomic DNA purified from one patient were independently analyzed at three laboratories. This study was approved by the Ethics Committee of Osaka City University Graduate School of Medicine and all blood samples were collected with written informed consent.
Cell lines and culture
Human 293T and 22Rv1 cells were obtained from the American Type Culture Collection (CRL-1537 and CRL-2525, respectively; ATCC, Manassas, VA). Human prostate cancer cell line LNCap-FGC was obtained from the RIKEN Cell Bank (Tukuba, Japan), and the GP293 packaging cell line was purchased from Clontech Laboratories (Mountain View, CA). These cells were grown in Dulbecco's modified essential medium supplemented with 10% fetal bovine serum (FBS) and antibiotics. Rat hybridoma cell line R187 was obtained from ATCC (CRL-1912) and maintained in RPMI-1640 medium supplemented with 50 nM 2-mercaptoethanol, 10% FBS, and antibiotics. Before collecting the culture supernatant, the growth medium was replaced with CD Hybridoma medium (Invitrogen, Carlsbad, CA) supplemented with 8 mM l-glutamine. For recombinant Env production, Sf9 and High Five cells (Invitrogen) were maintained in Sf-900 III SFM and Expressed Five medium (Invitrogen), respectively.
IgG proteins in culture supernatants from R187 cells, prepared against SFFV Gag and able to react with Gag capsid proteins from a wide variety of gammaretroviruses , were purified using a protein G affinity column (MabTrap Kit; Amersham Biosciences, Piscataway, NJ). For anti-Env Abs, rabbits were immunized with a mixture of two peptides (PRVPIGPNPV[C] of Env SU and [C]QFEQLAAIHTDLG of Env TM; [C] indicates an additional cysteine residue for peptide purification), and their antisera were collected and purified after five immunization steps with a Protein A affinity column (GE Healthcare, Buckinghamshire, UK). Concentrations of the purified R187 mAb and anti-Env pAb were 4.0 mg/ml and 8.5 mg/ml, respectively.
An infectious XMRV molecular clone, pcDNA3.1-VP62, was provided by Dr. R. H. Silverman. To produce the viral particles, 293T cells were transfected with pcDNA3.1-VP62 by a liposome method (Lipofectamine LTX; Invitrogen). Two days after transfection, the culture supernatant was collected, filtered, and concentrated 20 times by centrifugation at 20,000 × g for 4 h at 4°C. The concentrated virus was suspended in a Laemmli SDS sample buffer. As a negative control, we prepared an env-defective HIV-1 virus (pNL∆env, provided by Dr. A. Adachi) by using the same method as for XMRV. A MoMLV-derived retrovirus vector was produced using the GP293 cell line, with or without transfection of an amphotropic Env expression vector (provided by Dr. D. R. Littman). Viral proteins were separated by 5-20% gradient SDS-PAGE and either stained with SYPRO Ruby (Bio-Rad, Hercules, CA) or transferred to a polyvinylidene difluoride membrane (Wako Pure Chemical Industries, Osaka, Japan) cut into strips. After blocking with 5% skimmed milk in Tris-buffered saline (TBS), the strips were incubated with 1:100 diluted donor or patient plasma samples at 4°C overnight. After washing with TBS containing 0.05% Tween-20, the strips were incubated with 1:5,000 diluted horseradish peroxidase (HRP)-conjugated anti-human IgG Ab (GE Healthcare), and detected by ECL Plus (GE Healthcare). For endpoint dilutions, a pair of strips was blotted with 0.8 μg/ml-6.25 ng/ml (1:5,000-1: 640,000) R187 mAb and detected using 1:5,000 diluted HRP-conjugated anti-rat IgG (H+L) secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) for Gag, or blotted with 8.5 μg/ml-66.4 ng/ml (1:1,000-1:128,000) anti-Env pAb and detected using 1:2,500 diluted HRP-conjugated anti rabbit IgG (GE Healthcare).
Other immunoblot assays
To produce GST-fused XMRV Gag CA protein, a 789-bp fragment of the CA gene was amplified using genomic DNA of 293T cells infected with XMRV derived from 22Rv1 cells, and cloned into the pET-42b(+) vector (Merck KGaA, Darmstadt, Germany). The GST-CA protein was purified by a Glutathione-Sepharose 4B column (GE Healthcare) from bacterial lysate of BL21 Star (DE3) (Invitrogen) transformed by the GST-fused CA expression plasmid. To produce His-tagged recombinant Env SU of xenotropic MLV , a PCR-amplified env SU region was cloned into pcDNA3.1myc/His (Invitrogen) followed by subcloning of an env-His DNA fragment into the Bac-to-Bac Baculovirus Expression System (Invitrogen). The supernatant of Sf9 cells transfected with the bacmid was used for infection of HighFive cells. Recombinant Env proteins collected from the culture supernatant of infected cells were purified using a HisTrapHP column (GE Healthcare). In the native-PAGE, concentrated viruses were suspended with native sample buffer (Native Sample Buffer; Bio-Rad) and separated on a 5-20% gel in a Tris-glycine buffer (25 mM Tris-Cl, 192 mM glycine, pH 8.4). The subsequent procedures were for the Ab-screening immunoblot assay.
Detection of viral nucleic acids
For RT-PCR analysis of Ab-positive PC patient samples (Figure 4A), RNA was isolated from 500 μl of plasma using the PureLink Viral RNA/DNA Kit (Invitrogen), and 8 μl of the 10 μl eluted RNA was reverse-transcribed using Superscript III (Invitrogen) with random hexamer primers in a total reaction volume of 10 μl. In the nested PCR assay, 3 μl cDNA or 100 ng genomic DNA of PBMCs was amplified in a 20 μl volume with primer pairs GAG-O-F/R and GAG-I-F/R  and AmplyTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA) for 50 cycles. The PCR cycling conditions were as follows: activation at 95°C for 5 min; then 50 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 60 s (30 s in the second-round PCR); with a final extension at 72°C for 7 min.
To extract genomic DNA from CFS patients, 4 ml of whole blood in EDTA were centrifuged at 1500 × g for 10 min at room temperature, and 200 μl of the buffy coat were transferred to a 2 ml tube for DNA purification using the QIAamp Blood Mini Kit (Qiagen GmbH, Hilden, Germany). We divided 180 μl of eluted DNA equally into three tubes for analysis at three independent laboratories: Department of Research, Japanese Red Cross Osaka Blood Center, and the Laboratories of Signal Transduction and Viral Pathogenesis, Institute for Virus Research, Kyoto University, Japan. PCR of 1 μg genomic DNA in a 50 μl reaction was performed with primer pairs GAG-O-F/R and GAG-I-F/R  for nested genomic PCR (data not shown) or 419F and 1154R  and In-For363 and n-Rev536  for single PCR. In the genomic PCRs, we used PrimeSTAR GXL DNA polymerase (Takara Bio, Shiga, Japan) with the following conditions: activation at 98°C for 2 min; then 45 cycles of 98°C for 10 s, 63°C for 15 s, and 68°C for 45 s; and a final step at 68°C for 2 min. For one-step RT-PCR (Figure 5A), RNAs were purified from 1 ml of 4-day culture supernatants of P24 PBMCs activated with 10 ng/ml concanavaline A (J-Oil Mills, Tokyo, Japan) and 100 U/ml IL-2 (e-Bioscience, San Diego, CA) and maintained with LNCap-FGC cells or patient plasma using a QIAamp Ultrasense Virus Kit (Qiagen). One-step RT-PCR was performed using 15 μl of 60 μl eluted RNA and a 419F and 1154R primer pair  and the following conditions: reverse transcription at 50°C for 30 min; activation at 95°C for 15 min; then 45 cycles of 94°C for 30 s, 57°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 10 min.
TaqMan real-time PCR tests were performed with 200 ng of genomic DNA, Universal ProbeLibrary, and FastStart TaqMan Probe Master (Roche, Basel, Switzerland) in a total reaction volume of 20 μl with a Rotor-Gene Q thermal cycler (Qiagen). Primer and probe sequences are as follows: 5'-cctagtggccaccaaacaat-3' (Env forward), 5'-ggccccaaggtctgtatgta-3' (Env reverse), and 5'-FAM-gctccagg-3' (Env probe, #1 of Universal ProbeLibrary). The following condition was used: 1 cycle of 95°C for 10 min, and 50 cycles of 95°C for 15 s and 60°C for 45 s.
In patients whose serum tested positive for XMRV RNA, mutations of RNASEL at amino acid positions 462  and 541  were examined as previously described [1, 20]. PCR-amplified genomic DNA fragments were sequenced using an ABI PRISM 3100 genetic analyzer (Applied Biosystems).
Non-parametric analysis was performed with the Mann-Whitney U -test to determine any statistical significance in the data. A p value of less than 0.05 was considered to be significant.