XMRV/MLV infection is rare in prostate cancer¹^,².

1<p>Percentages in parentheses.</p>2<p>Dashes indicate test not performed on these sample types.</p>3<p>nPCR, nested PCR.</p

Contamination of commercial AMV reverse transcriptases (RT) with MLV sequences.

<p>A. Representative real-time, generic MLV protease (<i>pro</i>) amplification plot. Blue lines, XMRV RNA standard extracted from 22Rv1 cell culture supernatants from 10⁶–10⁰ copies per reaction; burgundy lines with triangles, 4/16 (25%) water only controls tested positive for MLV <i>pro</i> sequences using the ABI TaqMan Fast 1-step Master Mix; bright green line, RFU, relative fluorescent units. B. Representative gel image showing nested PCR detection of 208-bp MLV polymerase (<i>pol</i>) sequences in water only control reactions using Finnzymes RobustI AMV RT. Lanes 1–16, water only controls; lanes 17–20, XMRV RNA extracted from 22Rv1 cell culture supernatants from 10³, 10², and 10 copies per reaction, respectively; M. molecular weight marker.</p

Contamination of commercial RT-PCR reagents with murine leukemia virus (MLV).¹

1<p>The reverse transcriptase (RT) enzymes and kits were tested by quantitative protease (<i>pro</i>), polymerase (<i>pol</i>), <i>gag and</i>/or nested <i>pol</i> RT-PCR assays and results shown are number of positive tests out of the total number of replicates. Percentages for replicate testing and copies/reaction (rxn) are in parentheses.</p>2<p>Mouse DNA contamination was detected using mitochondrial DNA (mtDNA) and intracisternal A particle (IAP) PCR qPCR assays.</p>3<p>ND, not done.</p

Identification of XMRV sequences in prostate cancer patients.

<p>Representative nested <i>pol</i> PCR results using prostate tumor DNA. Lanes 1–24, prostate cancer patients, including patients 5956 (lane 8) and 6203 (lane 19); lane 25, negative human PBMC DNA control; lanes 26 and 27, water only controls for primary and nested PCR tests, respectively; lanes 28 and 29, assay sensitivity controls consisting of 10 and 10³ copies of XMRV VP62 plasmid DNA diluted in a background of 1 ug of human PBMC DNA, respectively.</p

Absence of antibodies to XMRV and MLV in prostate cancer patients.

<p>Molecular weight markers (kD) are provided on the left of the WBs in the upper panels. Expected sizes of viral Gag (p30, capsid (CA)), pr65, and Envelope (Env, gp69/71) proteins are provided in each WB in the upper panels. Representative WB results for eleven prostate cancer patients, including patients 5956 and 6203 (indicated with asterisks). Determination of MLV specific reactivity is determined by comparison of seroreactivity to xenotropic MLV-infected HeLa antigens and uninfected HeLa antigens in upper and lower panels, respectively. Ra, Rauscher MLV whole virus goat polyclonal antisera; Fr, Friend MLV Envelope (gp69/71) goat polyclonal antisera; pre-immune, goat sera prior to immunization.</p

Inference of contamination origin in commercial RT-PCR reagents and human genomic DNAs.

<p>Phylogenetic analysis of 168-bp polymerase (<i>pol</i>) sequences. Bootstrap values ≥60 are shown. New sequences from the current study are shown in red (RT contaminants) and blue (human genomic DNA contaminants). Sequence names in purple are those reported as contaminants from other human studies. GenBank accession numbers for prototypical murine leukemia virus (MLV) reference sequences are provided in parentheses. XMRV clades are collapsed for presentation only and include identical sequences from CFS-WPI-1106(GQ497344), CFS-WPI-1178(GQ497343), preXMRV2(FR871850), PC-VP35(DQ241301), PC-VP42(DQ241302), PC-VP62(DQ399707), 22Rv1/CWR-R1(FN692043) The 168-bp <i>pol</i> sequences are available from the authors upon request. GenBank does not accept sequences less than 200-bp in length. Sequences coded as XMRV VP and WPI are from prostate cancer (PC) and CFS patients, respectively. BD; blood donor sequences. Viral host receptor tropism is indicated by blue (xenotropic), purple (polytropic), and yellow (ecotropic) spheres. PMLV, polytropic MLV; XMLV, xenotropic MLV.</p

Detection of XMRV in three prostate cancer patients¹.

1<p>Dashes indicate test not performed.</p>2<p>PTT, prostate tumor tissue.</p>3<p>nPCR, nested PCR; <i>gag</i>, group specific antigen; <i>pol</i>, polymerase; <i>env</i>, envelope.</p>4<p>qRT-PCR, quantitative RNA PCR; <i>pro</i>, protease gene.</p>5<p>nRT-PCR, nested RNA PCR.</p>6<p>Murine PCR, test for detection of specimen contamination with mouse cells or DNA using mitochondrial primers (MCOX2).</p>7<p>For specimens 5935 and 5956 testing includes results of triplicates and the initial screening. Quantity of DNA for 6203 was insufficient for triplicate testing.</p

Identification of variant XMRV in prostate cancer patients using phylogenetic analysis.

<p>A. envelope (<i>env</i>), B. polymerase (<i>pol</i>), and C. <i>gag</i>. Stability of the tree topology was tested using 1000 bootstrap replicates in both neighbor joining (NJ) and maximum likelihood (ML) methods. Bootstrap values >60 are shown at major nodes (NJ/ML). New sequences from the current study are boxed. Accession numbers for prototypical MLV sequences available at GenBank are XMRV VP35 = DQ241301, XMRV VP62 = DQ399707, XMRV VP42 = DQ241302, XMRV WPI-1106 = GQ497344, XMRV WPI-1178 = GC497343, XMRV PCA1–PCA17 = GU812341–GU812357, MLV DG-75 = AF221065, MLV MTCR = NC_001702, MLV AKV = J01998, MLV BM5eco = AY252102.1, Moloney MLV = J02255, Moloney neuropthogenic MLV variant ts1-92b = AF462057, Rauscher MLV = NC_001819, Friend MLV = X02794, mERV Chr 7 = AC167978, mERV Chr 7 = AC127565, mERV Chr 8 = AC127575, mERV Chr 12 = AC153658, mERV Chr 9 = AC121813, mERV Chr 4 = AL627077, mERV Chr 1 = AC083892), XMLV A2780 = FR670594, XMLV BHY = FR670595, XMLV Daudi = FR670596, XMLV EKVX = FR670597, XMLV IMR-5 = FR670598, XMLV MUTZ-1 = FR670599, XMLV S-117 = FR670600, XMLV TYK-nu = FR670601. Sequences denoted RAW are from the polytropic MLV isolated in HeLa cells used to develop the in-house WB test. Sequences coded as XMRV VP and PCA and WPI are from prostate cancer and CFS patients, respectively. Additional prostate cancer patient VP <i>gag</i> and <i>pol</i> sequences were kindly provided by Drs. Robert Silverman and Joe Derisi. Viral tropism, as determined by analysis of <i>env</i> sequences, is indicated by blue (xenotropic), purple (polytropic), and yellow (ecotropic) spheres.</p

Contamination of commercial human DNAs with mouse DNA.¹

1<p>1 ug of the human DNAs were tested using quantitative (qPCR) <i>pro</i> and nested <i>pol</i> and <i>gag</i> PCR assays. Copies/µg DNA is provided in parentheses for specimens with evidence of contamination.</p>2<p>Mouse DNA contamination was detected using mitochondrial DNA (mtDNA) and intracisternal A particle (IAP) PCR qPCR assays.</p>3<p>Second lot of human DNA sample received from Biochain after informing them of the contamination results.</p>4<p>NA, information not available.</p

Inference of contamination origin in commercial human genomic DNAs.

<p>Phylogenetic analysis of 301-bp <i>gag</i> sequences. Bootstrap values ≥60 are shown. New sequences from the current study are shown in blue (human genomic DNA contaminants). Sequence names in purple are those reported as contaminants from other human studies. GenBank accession numbers for prototypical murine leukemia virus (MLV) reference sequences are provided in parentheses. XMRV clades are collapsed for presentation only and include CFS-WPI-1106(GQ497344), CFS-WPI-1178(GQ497343), CFS-WPI-1130(GQ483508), CFS-WPI-1138(GQ483509), CFS-WPI-1169(GQ483510), CFS-WPI-CI-1303(JF907633), CFS-WPI-CI-1313(JF907643), CFS-WPI-CI-1314T(JF907644), CFS-WPI-CI-1307(JF907638), CFS-WPI-CI-1310(JF907641), CFS-WPI-CI-1327(JF907636), preXMRV2(FR871850), PC-VP35(DQ241301),and PC-VP62(DQ399707). Accession numbers for the new <i>gag</i> sequences generated in our study are JN629081-JN629087. Sequences coded as XMRV VP and WPI are from prostate cancer (PC) and CFS patients, respectively. BD; blood donor sequences. Viral host receptor tropism is indicated by blue (xenotropic), purple (polytropic), and yellow (ecotropic) spheres. PMLV, polytropic MLV; XMLV, xenotropic MLV.</p