RESUMO
Murine leukemia viruses (MLVs), including xenotropic-MLV-related virus (XMRV), have been controversially linked to chronic fatigue syndrome (CFS). To explore this issue in greater depth, we compiled coded replicate samples of blood from 15 subjects previously reported to be XMRV/MLV-positive (14 with CFS) and from 15 healthy donors previously determined to be negative for the viruses. These samples were distributed in a blinded fashion to nine laboratories, which performed assays designed to detect XMRV/MLV nucleic acid, virus replication, and antibody. Only two laboratories reported evidence of XMRV/MLVs; however, replicate sample results showed disagreement, and reactivity was similar among CFS subjects and negative controls. These results indicate that current assays do not reproducibly detect XMRV/MLV in blood samples and that blood donor screening is not warranted.
Assuntos
Sangue/virologia , Síndrome de Fadiga Crônica/virologia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/isolamento & purificação , Anticorpos Antivirais/sangue , Coleta de Amostras Sanguíneas , Linhagem Celular , Técnicas de Cocultura , Reações Falso-Positivas , Feminino , Humanos , Laboratórios , Masculino , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Infecções por Retroviridae/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Viremia , Replicação Viral , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/genética , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/imunologia , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/fisiologiaRESUMO
In October 2009, we reported the first direct isolation of infectious xenotropic murine leukemia virus-related virus (XMRV). In that study, we used a combination of biological amplification and molecular enhancement techniques to detect XMRV in more than 75% of 101 patients with chronic fatigue syndrome (CFS). Since our report, controversy arose after the publication of several studies that failed to detect XMRV infection in their CFS patient populations. In this addenda, we further detail the multiple detection methods we used in order to observe XMRV infection in our CFS cohort. Our results indicate that PCR from DNA of unstimulated peripheral blood mononuclear cells is the least sensitive method for detection of XMRV in subjects' blood. We advocate the use of more than one type of assay in order to determine the frequency of XMRV infection in patient cohorts in future studies of the relevance of XMRV to human disease.
Assuntos
Células Sanguíneas/virologia , Síndrome de Fadiga Crônica/virologia , Reação em Cadeia da Polimerase/métodos , Virologia/métodos , Vírus Relacionado ao Vírus Xenotrópico da Leucemia Murina/isolamento & purificação , Humanos , Sensibilidade e EspecificidadeRESUMO
In 2006, sequences described as xenotropic murine leukemia virus-related virus (XMRV) were discovered in prostate cancer patients. In October 2009, we published the first direct isolation of infectious XMRV from humans and the detection of infectious XMRV in patients with chronic fatigue syndrome. In that study, a combination of classic retroviral methods were used including: DNA polymerase chain reaction and reverse transcriptase polymerase chain reaction for gag and env, full length genomic sequencing, immunoblotting for viral protein expression in activated peripheral blood mononuclear cells, passage of infectious virus in both plasma and peripheral blood mononuclear cells to indicator cell lines, and detection of antibodies to XMRV in plasma. A combination of these methods has since allowed us to confirm infection by XMRV in 85% of the 101 patients that were originally studied. Since 2009, seven studies, predominantly using DNA polymerase chain reaction of blood products or tumor tissue, have reported failures to detect XMRV infection in patients with either prostate cancer or chronic fatigue syndrome. A review of the current literature on XMRV supports the importance of applying multiple independent techniques in order to determine the presence of this virus. Detection methods based upon the biological and molecular amplification of XMRV, which is usually present at low levels in unstimulated blood cells and plasma, are more sensitive than assays for the virus by DNA polymerase chain reaction of unstimulated peripheral blood mononuclear cells. When we examined patient blood samples that had originally tested negative by DNA polymerase chain reaction by more sensitive methods, we observed that they were infected with XMRV; thus, the DNA polymerase chain reaction tests provided false negative results. Therefore, we conclude that molecular analyses using DNA from unstimulated peripheral blood mononuclear cells or from whole blood are not yet sufficient as stand-alone assays for the identification of XMRV-infected individuals. Complementary methods are reviewed, that if rigorously followed, will likely show a more accurate snapshot of the actual distribution of XMRV infection in humans.
Assuntos
Síndrome de Fadiga Crônica/virologia , Vírus da Leucemia Murina/isolamento & purificação , Neoplasias da Próstata/virologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/genética , Síndrome de Fadiga Crônica/genética , Genes env , Genes gag , Humanos , Vírus da Leucemia Murina/genética , Masculino , Neoplasias da Próstata/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções Tumorais por Vírus/genética , Infecções Tumorais por Vírus/virologiaRESUMO
Chronic fatigue syndrome (CFS) is a debilitating disease of unknown etiology that is estimated to affect 17 million people worldwide. Studying peripheral blood mononuclear cells (PBMCs) from CFS patients, we identified DNA from a human gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), in 68 of 101 patients (67%) as compared to 8 of 218 (3.7%) healthy controls. Cell culture experiments revealed that patient-derived XMRV is infectious and that both cell-associated and cell-free transmission of the virus are possible. Secondary viral infections were established in uninfected primary lymphocytes and indicator cell lines after their exposure to activated PBMCs, B cells, T cells, or plasma derived from CFS patients. These findings raise the possibility that XMRV may be a contributing factor in the pathogenesis of CFS.