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- Open Access
PCR and serology find no association between xenotropic murine leukemia virus-related virus (XMRV) and autism
© Satterfield et al; licensee BioMed Central Ltd. 2010
- Received: 14 May 2010
- Accepted: 14 October 2010
- Published: 14 October 2010
Xenotropic murine leukemia virus-related virus (XMRV) is a retrovirus implicated in prostate cancer and chronic fatigue syndrome (CFS). Press releases have suggested that it could contribute to autism spectrum disorder (ASD). In this study we used two PCR assays and one antibody assay to screen 25 blood samples from autistic children born to mothers with CFS and from 20 mixed controls including family members of the children assayed, people with fibromyalgia and people with chronic Lyme disease. Using a real-time PCR assay, we screened an additional 48 South Carolina autism disorder samples, 96 Italian ASD samples, 61 South Carolina ASD samples and 184 healthy controls. Despite having the ability to detect low copy number XMRV DNA in a large background of cellular DNA, none of the PCR assays found any evidence of XMRV infection in blood cells from patients or controls. Further, no anti-XMRV antibodies were detected, ruling out possible low level or abortive infections in blood or in other reservoirs. These results imply that XMRV is not associated with autism.
- Autism Spectrum Disorder
- Autism Spectrum Disorder
- Chronic Fatigue Syndrome
- Autistic Child
Xenotropic murine leukemia virus-related virus (XMRV) is a recently discovered retrovirus that can infect humans . Studies of the virus in prostate cancer have given conflicting results, with between 0% and 27% of prostate cancers suggested to be associated with XMRV [2–4]. More recently, Lombardi, et al showed that 67% of people with chronic fatigue syndrome (CFS) were positive for XMRV by PCR amplification of the gag gene . In an interview given on the same day as the Lombardi publication, Dr Mikovits stated that they had found XMRV in a 'significant number' of autism spectrum disorder (ASD) samples and speculated that 'this might even explain why vaccines lead to autism in some children' . Shortly thereafter, widely circulated articles appeared, containing non-peer reviewed data with reports that XMRV may be present in ≥40% of people with autism . Given the recent controversy over the connection between ASD and the MMR (measles, mumps, rubella) vaccine, a scientific evaluation of these statements is important [8, 9]. ASD comprises multiple cognitive and developmental disorders, including autistic disorder (AD), Asperger disorder and PDD-NOS (pervasive developmental disorder, not otherwise specified). Although ASD affects an estimated 1 in every 110 individuals, no consistent causes for the disease have been identified .
In this study, we screened for a link between XMRV and autism. First, we looked for XMRV in 25 blood samples from children with ASD born to mothers with CFS and in 20 samples from a mix of controls including healthy family members of the children, people with fibromyalgia and people with chronic Lyme disease. We also assayed genomic DNA from 96 Italian ASD samples, 48 South Carolina AD samples, 61 South Carolina ASD samples and 184 healthy controls for presence of XMRV DNA. The healthy controls were a mix of healthy male and female college students and newborns from South Carolina.
Real-time PCR primer and probe sequences for the pol xenotropic murine leukemia virus-related virus (XMRV) assay.
pol (2794 to 3062)
[6FAM] TGTTCCAGGGGGACTGGCAAGGTACCAccctgg [DABC]2,3
Samples were obtained from the autistic children of mothers with CFS and from banked, well-characterized autism samples at the Greenwood Genetic Center. Informed consent was obtained from all participants or legally authorized representatives and identifying information was removed from each sample. For the former cohort, blood was collected, and the buffy coat was immediately isolated for nucleic acid extraction (Blood DNA Minikit; Qiagen, Valencia, CA, USA). Plasma samples were frozen for later analysis. Extracted DNA was quantified on a spectrophotometer (Nanodrop; Thermo Scientific, Wilmington, DE, USA) and checked for integrity with a minimum 260:280 ratio of 1.8. DNA was diluted to 100 ng/μl. For the latter cohort and the healthy controls, DNA was extracted from blood using proteinase K lysis and high-salt precipitation as previously described . For the samples from autistic children born to mothers with CFS, 25 μl of sample (2.5 μg of DNA and 833 ng of peripheral blood mononuclear cell (PBMC) DNA) were added to 25 μl of a commercial master mix (Simplex DNA Master Mix; Cooperative Diagnostics, Greenwood, SC, USA) with a final primer concentration of 250 nmol/l and probe concentration of 200 nmol/l. The 25 ASD samples from children born to mothers with CFS and the 20 mixed control samples were also submitted to the Centers for Disease Control (CDC) for blinded assaying using an XMRV western blot and a nested PCR assay (external primers XPOLOF and XPOLOR; internal primers XPOLIF and XPOLIR as shown in the Switzer, et al publication) that detects both MuLV and XMRV pol gene sequences . The CDC nested PCR assay used 1.0 μg of DNA and was capable of detecting < 10 copies of VP62 XMRV DNA. The DNA integrity of the samples was verified by β-actin PCR as an internal control, as described previously . The western blot used denatured XMRV as antigen, which was crossreactive with anti-MuLV-positive goat sera for the envelope (glycoprotein (gp)69/71), transmembrane (p15E), matrix (p15), Gag (pr68) and capsid (p30) antigens. For the banked samples and healthy controls, 5 μl of sample (500 ng of DNA and 167 ng of PBMC DNA) were added to 5 μl of a master mix (Simplex DNA Master Mix; Cooperative Diagnostics) with primer and probe. Real-time PCR cycling conditions were 95°C for 20 seconds followed by 45 cycles of 95°C for 1 second and 60°C for 20 seconds. Two positive controls (10 pg of XMRV infected 22Rv1 cell DNA or 60 copies of VP62 XMRV synthetic DNA spiked into 2.5 μg of genomic DNA) and two negative controls (nuclease-free water) were included in each run.
Results of xenotropic murine leukemia virus-related virus (XMRV) real-time PCR.
Positives by assay1
Autistic children of mothers with CFS3
South Carolina autism disorder
South Carolina ASD
Newborn healthy controls
Male healthy controls
In detection of any new virus, false positives and false negatives are always a concern. In this case, the ability to detect low copy numbers of XMRV by two different PCR assays, both designed to recognize regions of XMRV/MuLV that are 100% conserved in all published XMRV strains, implies that the assays would be able to detect XMRV if it was present in the samples. It is possible that XMRV was present in the samples at levels below the detection limit of the PCR assays, but this seems unlikely given the relatively high frequency of infection found by Lombardi et al. in people with CFS using a much smaller amount of sample . The samples could have inhibited the PCR assay, but this is unlikely as XMRV DNA spiked into random patient samples was readily detected in every case. Further, no anti-XMRV antibodies were detected, precluding the possibility of low-level or abortive infections in blood or other reservoirs that the PCR assay might have missed.
The presence of false positives in the CFS-XMRV findings cannot be ruled out, and it is supported by the following observations. A previous attempt to validate the findings of Lombardi et al. found no XMRV in 186 CFS samples . A second independent attempt also failed to find any association between XMRV and CFS, using both PCR and XMRV antibody assays in the 170 CFS samples assayed . A third attempt in the Netherlands also failed to find XMRV in 32 CFS samples , and a fourth attempt in the US also failed to find XMRV in 51 CFS samples .
Another interesting differentiator between our data and previous results is the lack of evidence of PCR-detectable XMRV in the blood of healthy controls. The Lombardi et al. study showed that in addition to 67% of people with CFS having PCR-detectable levels of XMRV in the blood, there may be a baseline of infection within the general population that is as high as 4% . The three European studies, all of which failed to find evidence of XMRV in the blood of controls or CFS samples, seem to suggest that the 4% rate detected by Lombardi et al. is much greater than the actual prevalence in the world, and may have been inflated by either geographically confined assaying or false positives [14–16]. Our results confirm that suggestion; we found no evidence of XMRV in 204 controls and 230 autism samples (434 samples in total).
From the results presented here, if XMRV is present in people with autism, it is an extremely rare occurrence and is unlikely to be a significant contributor to the disorder.
We would like to acknowledge the Laboratory Branch of the Division of HIV/AIDS Prevention within the National Center for HIV/AIDS, Viral Hepatitis, STD and TB Prevention at the United States Centers for Disease Control for confirmatory testing with two PCR assays and the XMRV western blot.
- Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, Klein EA, Malathi K, Magi-Galluzzi C, Tubbs RR, Ganem D, Silverman RH, DeRisi JL: Identification of a novel gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog. 2006, 2: e25-10.1371/journal.ppat.0020025.PubMed CentralView ArticlePubMedGoogle Scholar
- Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR: XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors. Proc Natl Acad Sci USA. 2009, 106: 16351-16356. 10.1073/pnas.0906922106.PubMed CentralView ArticlePubMedGoogle Scholar
- Hohn O, Krause H, Barbarotto P, Niederstadt L, Beimforde N, Denner J, Miller K, Kurth R, Bannert N: Lack of evidence for xenotropic murine leukemia virus-related virus(XMRV) in German prostate cancer patients. Retrovirology. 2009, 6: 92-10.1186/1742-4690-6-92.PubMed CentralView ArticlePubMedGoogle Scholar
- Fischer N, Hellwinkel O, Schulz C, Chun FK, Huland H, Aepfelbacher M, Schlomm T: Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer. J Clin Virol. 2008, 43: 277-283. 10.1016/j.jcv.2008.04.016.View ArticlePubMedGoogle Scholar
- Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, Peterson DL, Ruscetti SK, Bagni RK, Petrow-Sadowski C, Gold B, Dean M, Silverman RH, Mikovits JA: Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science. 2009, 326: 585-589. 10.1126/science.1179052.View ArticlePubMedGoogle Scholar
- Mikovits JA, Whittemore A: Interview with Sam Shad. Nevada Newsmakers. 2009Google Scholar
- Kirby D: Is autism associated with a viral infection?. The Huffington Post. 2009, [http://www.huffingtonpost.com/david-kirby/is-autism-associated-with_b_316986.html]Google Scholar
- Retraction-Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children. Lancet. 1998, 375: 445-10.1016/S0140-6736(10)60175-4.Google Scholar
- Wakefield AJ, Murch SH, Anthony A, Linnell J, Casson DM, Malik M, Berelowitz M, Dhillon AP, Thomson MA, Harvey P, Valentine A, Davies SE, Walker-Smith JA: Ileal-lymphoid-nodular hyperplasia, non-specific colitis, and pervasive developmental disorder in children. Lancet. 1998, 351: 637-641. 10.1016/S0140-6736(97)11096-0.View ArticlePubMedGoogle Scholar
- Autism and Developmental Disabilities Monitoring Network Surveillance Year 2006 Principal Investigators; Centers for Disease Control and Prevention (CDC): Prevalence of autism spectrum disorders - Autism and Developmental Disabilities Monitoring Network, United States, 2006. MMWR Surveill Summ. 2009, 58: 1-20.Google Scholar
- Knouf EC, Metzger MJ, Mitchell PS, Arroyo JD, Chevillet JR, Tewari M, Miller AD: Multiple integrated copies and high-level production of the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) from 22Rv1 prostate carcinoma cells. J Virol. 2009, 83: 7353-7356. 10.1128/JVI.00546-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Schwartz CE, Ulmer J, Brown A, Pancoast I, Goodman HO, Stevenson RE: Allan-Herndon syndrome. II. Linkage to DNA markers in Xq21. Am J Hum Genet. 1990, 47: 454-458.PubMed CentralPubMedGoogle Scholar
- Switzer WM, Jia H, Hohn O, Zheng H, Tang S, Shankar A, Bannert N, Simmons G, Hendry RM, Falkenberg VR, Reeves WC, Heneine W: Absence of evidence of xenotropic murine retrovirus-related virus infection in persons with chronic fatigue syndrome and healthy controls from the United States. Retrovirology. 2010, 7: 57-10.1186/1742-4690-7-57.PubMed CentralView ArticlePubMedGoogle Scholar
- Erlwein O, Kaye S, McClure MO, Weber J, Wills G, Collier D, Wessely S, Cleare A: Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome. PLoS One. 5: e8519-10.1371/journal.pone.0008519.Google Scholar
- Groom HC, Boucherit VC, Makinson K, Randal E, Baptista S, Hagan S, Gow JW, Mattes FM, Breuer J, Kerr JR, Stoye JP, Bishop KN: Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome. Retrovirology. 2010, 7: 10-10.1186/1742-4690-7-10.PubMed CentralView ArticlePubMedGoogle Scholar
- van Kuppeveld FJ, de Jong AS, Lanke KH, Verhaegh GW, Melchers WJ, Swanink CM, Bleijenberg G, Netea MG, Galama JM, van der Meer JW: Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort. BMJ. 340: c1018-10.1136/bmj.c1018.Google Scholar
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