Autistic spectrum disorder (ASD) is characterized by impaired language, communication and social skills, as well as by repetitive and stereotypic patterns of behavior. Many autistic subjects display a dysregulation of the immune system which is compatible with an unresolved viral infection with prenatal onset, potentially due to vertical viral transmission. Recently, the xenotropic murine leukemia virus-related virus (XMRV) has been implicated in chronic fatigue syndrome (CFS) and in prostate cancer by several, though not all studies.
We assessed whether XMRV or other murine leukemia virus (MLV)-related viruses are involved in autistic disorder. Using nested PCR targeted to gag genomic sequences, we screened DNA samples from: (i) peripheral blood of 102 ASD patients and 97 controls, (ii) post-mortem brain samples of 20 ASD patients and 17 sex- and age-matched controls, (iii) semen samples of 11 fathers of ASD children, 25 infertile individuals and 7 fertile controls. No XMRV gag DNA sequences were detected, whereas peripheral blood samples of 3/97 (3.1%) controls were positive for MLV.
Citation: Lintas C, Guidi F, Manzi B, Mancini A, Curatolo P, Persico AM (2011) Lack of Infection with XMRV or Other MLV-Related Viruses in Blood, Post-Mortem Brains and Paternal Gametes of Autistic Individuals. PLoS ONE 6(2): e16609. doi:10.1371/journal.pone.0016609
Editor: Reuben Harris, University of Minnesota, United States of America
Received: December 1, 2010; Accepted: December 28, 2010; Published: February 23, 2011
Copyright: © 2011 Lintas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Italian Ministry for University, Scientific Research and Technology (P.R.I.N. n.2006058195 and 2008BACT54), the Italian Ministry of Health (RFPS-2007-5-640174), the Fondazione Gaetano e Mafalda Luce (Milan, Italy), a Trailblazer Award by Autism Speaks (Princeton, N.J.), and the Autism Research Institute (San Diego, CA). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder, characterized by different levels of impairment in social interaction and communication, as well as by stereotypies and rigid patterns of behaviour . Disease onset occurs prior to 3 years of age and its incidence is currently estimated at 1/150 live births –. ASD is the most heritable neuropsychiatric disorder, yet very few cases can be solely explained on the basis of de novo genetic mutations or cytogenetic abnormalities . Vertical viral transmission represents a non-genetic mechanism compatible with high parent-to-offspring transmission and with low rates of disease-specific genetic abnormalities . Clinically, many ASD patients display a dysregulation of the immune system, potentially suggestive of a prenatal-onset, unresolved viral infection –. Vertically transmitted viruses should be found more frequently in the affected tissues of autistic individuals compared to controls: based on this hypothesis we initially assessed the prevalence of several neurotropic viruses in post-mortem brains of autistic patients and controls, finding a significant association between ASD and polyomavirus infection . In the present study, we focus our attention on xenotropic murine leukemia virus-related virus (XMRV) and other xenotropic murine leukemia (MLV)-related viruses. These retroviruses indeed represent good candidates for vertical viral transmission in autism, because of their ability to integrate into the parental host genome and thus undergo parent-to-child transmission. Furthermore, XMRV infection is currently a source of serious concern in the USA for its possible link with chronic fatigue syndrome (CFS) .
Using nested PCR, XMRV and MLV gag genomic sequences were sought in the following biological samples: (a) peripheral blood mononuclear cells (PBMC) belonging to 102 ASD patients and 97 controls, (b) post-mortem brains of 20 ASD patients and 17 sex- and age-matched controls, and (c) semen samples belonging to 11 fathers of ASD children, 25 infertile individuals and 7 fertile controls. Our results do not support the frequent involvement of XMRV or MLV-related viruses in autism pathogenesis.
Patients and samples
All subjects, except for post-mortem brain donors, were recruited in Italy and are ethnically Italian. The demographic characteristics of these samples are summarized in Table 1. Briefly, (a) PBMC were obtained drawing blood from ASD patients diagnosed for any ASD (either Autistic Disorder, Asperger Disorder, or Pervasive Developmental Disorder Not Otherwise Specified) according to DSM-IV criteria , and clinically assessed as described . Controls were drawn as prescribed by family practitioner for a broad range of physical complaints unrelated to psychiatric disorders and among nursing and medical students at University Campus Biomedico (Rome, Italy), as described ; (b) frozen post-mortem brain tissues dissected from the superior temporal gyrus (Brodmann Areas 41/42 or 22) were obtained through the Autism Tissue Program from the NICHD Brain & Tissue Bank (Baltimore, MD) and the Harvard Brain Tissue Resource Center (Belmont, MA). These tissue samples largely overlap with those employed in our previous studies , as this neocortical region hosts well-documented structural and functional abnormalities in autism ; (c) semen samples were provided by outpatients who underwent andrological evaluation for infertility at the Division of Endocrinology of Catholic University of the Sacred Heart (U.C.S.C., Rome, Italy) upon vibratory stimulation using Ferticare Clinic (Multicept, Frederiksberg, Denmark), according to the ethical guidelines approved by the Institutional Review Boards (IRB) of Catholic University of the Sacred Heart and University Campus Bio-Medico (U.C.B.M.). Within 1 hour from collection, semen specimens were separated into seminal fluid and three cellular fractions (mobile sperm cells, sperm cells with hypomotility, and non-mobile cells including immobile spermatozoa, immature forms, leukocytes and epithelial cells) by centrifugation at 300 g for 30 minutes using Isolite® (IrvineScientific, Santa Ana, CA, USA); aliquots were stored at −80°C until DNA extraction. The consent forms signed by all individuals involved in blood and semen collection, including parents for their children, were approved by the Institutional Review Board of University Campus Bio-Medico (U.C.B.M.).
Nested PCR and sequencing
DNA was recovered by phenol/chloroform extraction and ethanol precipitation, following cell digestion with proteinase K at 55°C overnight. XMRV gag nested PCR was performed as previously described  with the following modifications: approximately 80 ng of genomic DNA in 25 µl final PCR reaction volume were used as a template for the first round PCR; 40 cycles were done for each round of amplification. In our hands, nested PCR sensitivity was at 10 viral copies, as in previous reports . Each PCR experiment included equal numbers of patients and controls, as well as negative controls for the first and second round PCRs; in order to minimize the risk of contaminations, positive controls were PCR-amplified separately and run on the same agarose for band size determination, as in our previous studies . Appropriately-sized PCR products (413 bp) were sequenced, using a CEQ8000 DNA sequencer (Beckman-Coulter, Fullerton, CA). In order to exclude contaminations with mouse genomic DNA, MLV positive samples were also assessed by a nested PCR targeting mouse histone deacetylase 5 (Hdac5) and displaying the same sensitivity as the nested PCR used to detect MLV.
No MLV-related virus gag sequences were detected in 96 blood samples and 20 post-mortem brains of ASD patients, as well as in 25 semen fractions belonging to 9 fathers of ASD children (Table 1). Similarly, 17 control brains, and 85 semen fractions from 7 fertile and 25 infertile controls (Table 1) were negative for MLV-related gag sequences. Three out of 97 (3.1%) peripheral blood samples from unaffected controls were positive (Figure 1). The difference between ASD blood samples and unaffected controls does not reach statistical significance (Fisher's exact P-value = 0.25, n.s.). DNA sequencing and BLAST analysis unveiled in 3 control blood samples viral gag gene sequences displaying 100% alignment with the mouse endogenous retrovirus MLV on chromosome 8 (GenBank Acc number: AC163617 nt 85467–85880). No mouse genomic contamination was detected in these three positive MLV control samples by nested PCR targeting the mouse histone deacetylase 5 (Hdac5) gene.
Our results show that infection with XMRV or other MLV-related viruses, assessed both in the central nervous system and in blood, is not associated with ASD nor is likely implicated in vertical viral transmission through parental gametes. We thus replicate and largely extend a recent study reporting no association between XMRV infection and autistic disorder .
A search for viruses as primary etiological agents in autism is well justified. Congenital infection with rubella or cytomegalovirus (CMV) represents one of the best-documented environmental factors significantly associated with ASD (for review see , ). The largest longitudinal study involving several hundred children prenatally exposed to rubella virus estimates at 7.4% the rate of autism in this group, much higher than ASD prevalence rates in the general population; risk appears especially high if rubella infection occurs during the first 8 weeks postconception –. Evidence linking prenatal CMV infection to autism is more circumstantial, but several case reports have been published –. Risk estimates are essentially based on a small cohort of 7 prenatally CMV-infected children, who displayed autistic features in 2 cases (2/7 = 28.6%) .
XMRV represents an interesting candidate to potentially play a role in autism pathogenesis. It was initially identified by PCR in approximately 10% of prostate cancer patients . It is phylogenetically related to MLV-related viruses and displays about 90% sequence identity with MLV . Recently, XMRV infection has been strongly associated with CFS . Attempts to replicate these initial results in European and North-American cohorts of prostate cancer and CFS patients have yielded conflicting results. In general, the association between XMRV infection and human disease appears stronger in the USA compared to Europe (Table 2). However, also four US studies are completely negative , , , , accounting for about two thirds of the total patient sample recruited in North America (Table 2). The discrepancy between European and North American studies could therefore reflect differences in PCR-based assay sensitivity rather than real geographical differences in the prevalence of infection by XMRV or other MLV-related viruses. In this respect, it will be important to establish and validate universal assays, as recently proposed by the National Institutes of Health.
Our results, combined with those reported by Sutherfield et al , render XMRV contributions to autism highly unlikely. Nonetheless we cannot exclude that MLV-related viruses may play a role in rare cases.
The authors gratefully acknowledge all the children and families who participated in this study. Post-mortem tissues were provided by the NICHD Brain & Tissue Bank (Baltimore, MD) and the Harvard Brain Tissue Resource Center (Belmont, MA), through the Autism Tissue Program (Princeton, NJ).
Conceived and designed the experiments: AMP CL. Performed the experiments: CL FG. Analyzed the data: AMP CL. Contributed reagents/materials/analysis tools: AMP AM PC BM. Wrote the paper: CL AMP.
- 1. American PsychiatricAssociation (2004) Diagnostic and statistical manual of mental disorders, 4th edn, 2004. Washington DC: American Psychiatric Press.
- 2. Fombonne E (2009) Epidemiology of pervasive developmental disorders. Pediatr Res 65: 591–598.
- 3. Rutter M (2005) Incidence of autism spectrum disorders: changes over time and their meaning. Acta Paediatr 94: 2–15.
- 4. Lintas C, Persico AM (2009) Autistic phenotypes and genetic testing: state-of-the-art for the clinical geneticist. J Med Genet 46: 1–8.
- 5. Persico AM (2010) Polyomaviruses and autism: more than simple association? J Neurovirol. Epub ahead of print.
- 6. Vargas DL, Nascimbene C, Krishnan C, Zimmerman AW, Pardo CA (2005) Neuroglial activation and neuroinflammation in the brain of patients with autism. Ann Neurol 57: 67–81.
- 7. Garbett K, Ebert PJ, Mitchell A, Lintas C, Manzi B, et al. (2008) Immune transcriptome alterations in the temporal cortex of subjects with autism. Neurobiol Dis 30: 303–11.
- 8. Lintas C, Altieri L, Lombardi F, Sacco R, Persico AM (2010) Association of autism with polyomavirus infection in postmortem brains. J Neurovirol 16: 141–9.
- 9. Lombardi VC, Ruscetti FW, Das Gupta J, Pfost MA, Hagen KS, et al. (2009) Detection of an infectious retrovirus, XMRV, in blood cells of patients with chronic fatigue syndrome. Science 326: 585–9.
- 10. Lintas C, Sacco R, Garbett K, Mirnics K, Militerni R, et al. (2009) Involvement of the PRKCB1 gene in autistic disorder: significant genetic association and reduced neocortical gene expression. Mol Psychiatry 14: 705–18.
- 11. Zilbovicius M, Meresse I, Chabane N, Brunelle F, Samson Y, et al. (2006) Autism, the superior temporal sulcus and social perception. Trends Neurosci 29: 359–66.
- 12. Urisman A, Molinaro RJ, Fischer N, Plummer SJ, Casey G, et al. (2006) Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant. PLoS Pathog 2: e25.
- 13. Switzer WM, Jia H, Hohn O, Zheng H, Tang S, et al. (2010) Absence of evidence of xenotropic murine leukemia virus-related virus infection in persons with chronic fatigue syndrome and healthy controls in the United States. Retrovirology 7: 57.
- 14. Satterfield BC, Garcia RA, Gurrieri F, Schwartz CE (2010) PCR and serology find no association between xenotropic murine leukemia virus-related virus (XMRV) and autism. Mol Autism 1: 14.
- 15. Libbey JE, Sweeten TL, McMahon WM, Fujinami RS (2005) Autistic disorder and viral infections. J Neurovirol 1: 1–10.
- 16. van den Pol AN (2006) Viral infections in the developing and mature brain. Trends in Neurosci 29: 398–406.
- 17. Chess S (1971) Autism in children with congenital rubella. J Autism Child Schizophr 1: 33–47.
- 18. Chess S (1977) Follow-up report on autism in congenital rubella. J Autism Child Schizophr 7: 69–81.
- 19. Chess S, Fernandez P, Korn S (1978) Behavioral consequences of congenital rubella. J Pediatr 93: 699–703.
- 20. Banatvala JE, Brown DW (2004) Rubella. Lancet 363: 1127–1137.
- 21. Stubbs EG (1978) Autistic symptoms in a child with congenital cytomegalovirus infection. J Autism Child Schizophr 8: 37–43.
- 22. Markowitz PI (1983) Autism in a child with congenital cytomegalovirus infection. J Autism Dev Disord 13: 249–253.
- 23. Stubbs EG, Ash E, Williams CP (1984) Autism and congenital cytomegalovirus. J Autism Dev Disord 14: 183–189.
- 24. Ivarsson SA, Bjerre I, Vegfors P, Ahlfors K (1990) Autism as one of several disabilities in two children with congenital cytomegalovirus infection. Neuropediatrics 21: 102–103.
- 25. Yamashita Y, Fujimoto C, Nakajima E, Isagai T, Matsuishi T (2003) Possible association between congenital cytomegalovirus infection and autistic disorder. J Autism Dev Disord 33: 455–459.
- 26. Sweeten TL, Posey DJ, McDougle CJ (2004) Brief report: autistic disorder in three children with cytomegalovirus infection. J Autism Dev Disord 34: 583–586.
- 27. López-Pisón J, Rubio-Rubio R, Ureña-Hornos T, Omeñaca-Teres M, Sans A, et al. (2005) Retrospective diagnosis of congenital infection by cytomegalovirus in the case of one infant. Rev Neurol 40: 733–736.
- 28. Kawatani M, Nakai A, Okuno T, Kobata R, Moriuchi M, et al. (2010) Detection of cytomegalovirus in preserved umbilical cord from a boy with autistic disorder. Pediatr Int 52: 304–307.
- 29. Henrich TX, Li JX, Felsenstein D, Kotton CX, Plenge RX, et al. (2010) Xenotropic Murine Leukemia Virus–Related Virus Prevalence in Patients with Chronic Fatigue Syndrome or Chronic Immunomodulatory Conditions. J Infect Dis. Oct 11. Epub ahead of print.
- 30. Aloia AL, Sfanos KS, Isaacs WB, Zheng Q, Maldarelli F, et al. (2010) XMRV: A New Virus in Prostate Cancer? Cancer Res. Oct 21. Epub ahead of print.
- 31. Danielson BX, Ayala GX, Kimata JX (2010) Detection of Xenotropic Murine Leukemia Virus–Related Virus in Normal and Tumor Tissue of Patients from the Southern United States with Prostate Cancer Is Dependent on Specific Polymerase Chain Reaction Conditions. J Infect Dis. Sep 3. Epub ahead of print.
- 32. Schlaberg R, Choe DJ, Brown KR, Thaker HM, Singh IR (2009) XMRV is present in malignant prostatic epithelium and is associated with prostate cancer, especially high-grade tumors. Proc Natl Acad Sci U S A 106: 16351–6.
- 33. Lo SC, Pripuzova N, Li B, Komaroff AL, Hung GC, et al. (2010) Detection of MLV-related virus gene sequences in blood of patients with chronic fatiguesyndrome and healthy blood donors. Proc Natl Acad Sci U S A 107: 15874–9.
- 34. van Kuppeveld FJ, de Jong AS, Lanke KH, Verhaegh GW, Melchers WJ, et al. (2010) 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.
- 35. Groom HC, Boucherit VC, Makinson K, Randal E, Baptista S, et al. (2010) Absence of xenotropic murine leukaemia virus-related virus in UK patients with chronic fatigue syndrome. Retrovirology 7: 10.
- 36. Verhaegh GW, de Jong AS, Smit FP, Jannink SA, Melchers WJ, et al. (2010) Prevalence of human xenotropic murine leukemia virus-related gammaretrovirus (XMRV) in dutch prostate cancer patients. Prostate. Sep 28. Epub ahead of print.
- 37. Erlwein O, Kaye S, McClure MO, Weber J, Wills G, et al. (2010) Failure to detect the novel retrovirus XMRV in chronic fatigue syndrome. PLoS One 6: e8519.
- 38. Fischer N, Hellwinkel O, Schulz C, Chun FK, Huland H, et al. (2008) Prevalence of human gammaretrovirus XMRV in sporadic prostate cancer. J Clin Virol 43: 277–83.
- 39. Hohn O, Krause H, Barbarotto P, Niederstadt L, Beimforde N, et al. (2009) Lack of evidence for xenotropic murine leukaemia virus-related virus (XMRV) in German prostate cancer patients. Retrovirology 6: 92.
- 40. Hong P, Li J, Li Y (2010) Failure to detect Xenotropic murine leukaemia virus-related virus in Chinese patients with chronic fatigue syndrome. Virol J 7: 224.
- 41. Cornelissen M, Zorgdrager F, Blom P, Jurriaans S, Repping S, et al. (2010) Lack of detection of XMRV in seminal plasma from HIV-1 infected men in The Netherlands. PLoS One 10: e12040.
- 42. Jeziorski E, Foulongne V, Ludwig C, Louhaem D, Chiocchia G, et al. (2010) No evidence for XMRV association in pediatric idiopathic diseases in France. Retrovirology 7: 63.
- 43. Fischer N, Schulz C, Stieler K, Hohn O, Lange C, et al. (2010) Xenotropic murine leukemia virus-related gammaretrovirus in respiratory tract. Emerg Infect Dis 16: 1000–2.
- 44. Barnes E, Flanagan P, Brown A, Robinson N, Brown H, et al. (2010) Failure to Detect Xenotropic Murine Leukemia Virus–Related Virus in Blood of Individuals at High Risk of Blood-Borne Viral Infections. J Infect Dis. Oct 11. Epub ahead of print.