Clin Microbiol Infect 2012; 18: 184–188
The human pathogen xenotropic murine leukaemia virus-related virus (XMRV) has been tentatively associated with prostate cancer and chronic fatigue syndrome. Unfortunately, subsequent studies failed to identify the virus in various clinical settings. To determine whether XMRV circulates in humans and the relationship with its host, we searched for the virus in 124 human immunodeficiency virus-infected patients who might have been exposed to XMRV, might be prone to infection as a result of progressive immunodeficiency, and had not yet been treated with antiretroviral drugs. Using nested PCR and single-step TaqMan real-time PCR, both designed on the XMRV gag gene, we could not find any positive samples. These findings add to the growing amount of scepticism regarding XMRV.
Xenotropic murine leukaemia virus-related virus (XMRV) is a novel gamma retrovirus of humans that was first discovered in 2006 . Originally, the virus was isolated from biopsy specimens of prostate cancer patients, and was believed to play an aetiological role in that disease; however, subsequent studies failed to reveal the presence of XMRV in large numbers of patients with prostate cancer, possibly because of uneven geographical distribution of the infection and/or differences in the sensitivity of the PCR methods used for detection [2–8]. In the past 4 years, several reports have documented the presence of XMRV in blood and respiratory secretions of subjects with chronic fatigue syndrome, adults with respiratory illness and transplant patients at rates ranging from 2% to 86%, suggesting that the virus is associated with various human diseases or is an endogenous virus that may take advantage of chronic inflammation processes or other underlying diseases (Table 1) [9–17]. To date, however, the aetiological role of XMRV, if any, is far from being proven. Moreover, the finding that tissues and biological fluids of healthy subjects also carry XMRV has led to scepticism regarding the real pathogenicity of the virus [5,6,9–11,13,15,16,18].
|Disease||No. of samples||Prevalence (%)||Method||Ethnicity||References|
|HIV-positive||124||0||Nested PCR and real-time PCR for gag||Italy||Present study|
|Prostate cancer (biopsies)||800||0||Real-time PCR for gag for and immunohistochemistry for Gag and Env cleavage products||USA||Aloia et al. |
|86||40 (RNase L QQ) |
1.5 (RNase L RQ)
|Real-time PCR for gag||USA||Urisman et al. |
|74||4||Real-time PCR||The Netherlands||Verhaegh et al. |
|589||0||Nested PCR and RT-PCR||Germany||Hohn et al. |
|334||6–23||Real-time PCR and immunohistochemistry||USA||Schlaberg et al. |
|105||1||Nested PCR||Germany||Fischer et al. |
|144||22||Nested PCR for env||USA||Danielson et al. |
|Chronic fatigue syndrome||39||Gag- and Env-ELISA assays; nested PCR||Germany||Hohn et al. |
|170||0||Real-time PCR||UK||Groom et al. |
|37||86.5||Nested PCR for gag||USA||Lo et al. |
|76||0||Real-time PCR assay for integrase gene and/or a nested PCR for gag||The Netherlands||van Kuppeveld et al. |
|65||0||Multiplex real-time PCR and RT-PCR||China||Hong et al. |
|102||0||Western blot, ELISA; gag and pol nested PCR||USA||Switzer et al. |
|101||75||Mikovits et al. |
|186||0||Nested PCR||UK||Erlwein et al. |
|32||0||Nested PCR assay for gag||USA||Henrich et al. |
|101||67||Nested PCR assay for gag||USA||Lombardi et al. |
|Multiple sclerosis with fatigue symptoms||112||0||Gag- and Env-ELISA assays; nested PCR||Germany||Hohn et al. |
|Autistic spectrum disorders||230||0||Real-time PCR and serology||Italy and South Carolina||Satterfield et al. |
|HCV-positive||67||0||PCR for gag or env||United Kingdom||Barnes et al. |
|HIV-positive||101 (acute) |
|0||PCR for gag or env||Switzerland and Spain||Barnes et al. |
|43||0||Nested PCR assay for gag||USA||Henrich et al. |
|199 plasmas |
50 culture supernatants
|0||Nested PCR or RT-PCR and real-time PCR assays||Cameroon and Uganda||Tang et al. |
|54 (seminal plasma)||0||Nested PCR for gag||The Netherlands||Cornelissen et al. |
|Rheumatoid arthritis||97||0||Nested PCR assay for gag||USA||Henrich et al. |
|Spondyloarthritis||19||0||Nested PCR for env||France||Jeziorski et al. |
|Paediatric haematological, neurological or inflammatory pathologies||62||0||Nested PCR for env||France||Jeziorski et al. |
|Paediatric respiratory diseases (nasopharyngeal aspirates)||80||0||Nested PCR for env||France||Jeziorski et al. |
|Respiratory tract infection without underlying disease||42 (sputum, nasal swab)||2.3||Nested PCR or real-time PCR||Germany||Fischer et al. |
|Respiratory tract infection without underlying COPD||31 (BAL)||3.2||Nested PCR or real-time PCR||Germany||Fischer et al. |
|Haematopoietic stem cell or solid organ transplant||161 (BAL, TS)||9.9||Nested PCR or real-time PCR||Germany||Fischer et al. |
|26||0||Nested PCR assay for gag||USA||Henrich et al. |
|Patients presenting for medical care||95||0||Nested PCR assay for gag||USA||Henrich et al. |
|Healthy controls||40||0||gag- and env-ELISA assays; nested PCR||Germany||Hohn et al. |
|70 (prostate biopsy)||1.3||Nested RT-PCR||Germany||Fischer et al. |
|62 (BAL, throat swab)||3.2||Nested PCR or real-time PCR||Germany||Fischer et al. |
|97||0||Western blot, ELISA; gag and pol nested PCR||USA||Switzer et al. |
|44||6.8||Nested PCR for gag||USA||Lo et al. |
|218||3.7||Nested PCR for gag||USA||Lombardi et al. |
|395||0||Quantitative PCR||UK||Groom et al. |
|204||0||Real-time PCR and serology||Italy and USA||Satterfield et al. |
|65||0||Multiplex real-time PCR and RT-PCR||China||Hong et al. |
Many aspects of the natural history and pathogenesis of XMRV are still poorly understood . These include the prevalence in the general population and in categories of subjects who may have greater chances of acquiring XMRV infection, and the relationship that the virus establishes with the infected host. Among these individuals, those with human immunodeficiency virus (HIV) infection are particularly at risk, as they have most likely been exposed to other infectious agents, and, if acquired, XMRV could have taken advantage of the progressive immunodeficiency in order to establish a persistent infection in the host.
We studied a cohort of 124 HIV-positive patients who had never received antiretroviral therapy. The presence of XMRV infection in peripheral blood was investigated by using two independent molecular methods. Furthermore, in light of the conflicting evidence on the appropriateness of investigated cohorts and methods used to look for the virus, we reviewed previous studies showing the advantages and disadvantages of the methodology used.
Materials and Methods
Patients and samples
The study group consisted of 124 randomly selected patients (97 males and 27 females; mean age 47 ± 14 years; range 18–80 years). All of them were HIV-seropositive as assessed by commercial immunoenzymatic and immunoblotting assays, and HIV-viraemic as assessed by commercial molecular assays. All patients were treatment-naive. After informed consent had been obtained, peripheral blood was sampled in EDTA tubes from each patient. CD4+ T-lymphocyte counts in peripheral blood at the time of sampling were recorded for each patient. Aliquots of whole blood and plasma were immediately prepared, stored at −80°C, and kept frozen until use.
XMRV infection was assessed by extracting DNA from 400 μL of whole blood with the Maxwell 16 System, according to the manufacturer’s instructions (Promega, Madison, WI, USA), using 500–750 ng of DNA as PCR template. The presence of virus was determined by using the protocol described in part by Lombardi . This includes a nested PCR and single-step TaqMan real-time PCR, both designed on XMRV gag. In the nested PCR, a 413-bp fragment was amplified in the first PCR. This was carried out for 35 cycles with sense primer GAG-O-F (5′-CGCGTTCGATTTGTTTTGTT-3′) and antisense primer GAG-O-R (5′-CCGCCTCTTCTTCATTGTTC-3′), under the following conditions: melting at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 45 s. The product of this reaction was then reamplified for 35 cycles with internal primers GAG-I-F (5′-TCTCGAGATCATGGGACAGA-3′) and GAG-1-R (5′-AGAGGGTAAGGGCAGGGTAA-3′) under the same PCR conditions except for 54°C for annealing. The reactions were carried out in a 50-μL PCR mixture containing Taq DNA polymerase, each dNTP at a concentration of 12.5 mM, primers (20 pmol/μL each), and optimized buffer components. All samples were tested at least in duplicate and on different occasions. The amplified product was analysed by electrophoresis on an agarose gel after ethidium bromide staining. Amplicon size was compared with standard molecular size markers. XMRV presence was also evaluated by a single-step TaqMan real-time PCR, designed on a 83-nucleotide fragment of gag and recently developed in our laboratory. Briefly, the assay was performed in a 25-μL format containing 0.9 μM each primer (Q445T, 5′-GGACTTTTTGGAGTGGCTTTGTT-3′; Q528R, 5′-GCGTAAAACCGAAAGCAAAAAT-3′), 0.1 μM labelled probe (F480PRO: FAM-ACAGAGACACTTCCCGCCCCCG-BHQ), 5 μL of extracted viral DNA, and a Master Mix containing dNTPs and Taq DNA polymerase. The reaction was run in triplicate for each sample in a iQCYCLER real-time PCR detection instrument (Bio-Rad Laboratories, Hercules, CA, USA), with a previously standardized program (95°C for 10 min; 55 cycles of 95°C for 15 s, and 60°C for 60 s). PCR data were collected and analysed with the iQ5 optical system software, version 2.1, developed by Bio-Rad. To exclude carryover contamination, negative controls were added during the DNA extraction and PCR amplification steps. To validate the amplification process, positive controls obtained from supernatants of a constitutively XMRV-infected B-cell line were run in each PCR. The assay sensitivities were measured by testing quadruplicates of ten-fold dilutions of the XMRV-positive control. The real-time PCR was found to have a sensitivity (about 100 DNA copies per millilitre of whole blood) of at least a dilution higher than that of nested PCR.
HIV viraemia ranged between 21 and 5 620 000 RNA copies per millilitre of plasma, with a median of 253 log10, revealing that plasma HIV load was relatively low in the majority of patients. The median level of CD4+ T-lymphocytes was 331 cells/μL: 36 (29%) subjects showed CD4+ T-lymphocyte counts below 200 cells/μL, whereas in 28 patients, the absolute numbers of CD4+ T-lymphocytes were within normal values for people of the same age (e.g. 500–1500 cells/μL). XMRV infection was first examined by nested PCR. No patient had detectable viral DNA in the blood (Table 1). As the sensitivity of nested PCR is lower than that of real-time PCR, which became available after the study commenced, all patients were retested with the latter method. Again, no evidence of XMRV DNA could be found, suggesting that this novel retrovirus was not present in whole blood of study patients.
The existence and clinical significance of XMRV is still disputed . Although the virus was initially isolated from patients with prostate cancer and CFS, subsequent findings have failed to unequivocally link XMRV to these diseases. To further complicate this picture, it has been recently suggested that XMRV detected in patient samples is probably the result of PCR contamination with mouse DNA, and that the tumour cell line 22Rv1, from which the original XMRV clone was isolated and characterized, was probably infected by murine XMRV during xenografting in mice [20,21]. Supporting this idea is the very high similarity between XMRV and its murine counterpart. As compared with the latter, however, XMRV has a 24-bp deletion within the gag leader sequence , which is the genomic region targeted by most molecular methods developed to detect the XMRV genome. This leaves room for alternative hypotheses . Supporting the exogenous origin is the observation that a few human tissues do not to express the human innate antiviral resistance factor APOBEC3G, and may thus create favourable conditions for XMRV replication and dissemination in the host . These facts and the lack of identification of XMRV or XMRV-related sequences in the overwhelming majority of studies published so far (Table 1) strengthen the hypothesis that XMRV is a zoonosis rather than a genuine human pathogen. Whatever the origin, it is necessary to determine cell types and organs that support viral replication and persistence in order to define the clinical significance of XMRV in humans. Although the prevalence of XMRV infection has been extensively investigated in various clinical settings, definition of target tissue(s) is far from being completed. In this study, the presence of XMRV DNA in peripheral blood samples of 124 patients with HIV infection was investigated with two molecular assays. All blood samples were XMRV DNA-negative, indicating that the virus is either absent from the peripheral circulating cells of HIV-infected individuals or is very rare. Although the presence of XMRV at levels below our detection threshold cannot be ruled out, this is very unlikely, as all samples were tested in duplicate or triplicate with two highly sensitive amplification methods (i.e. nested PCR and real-time PCR), both designed on the basis of highly conserved fragments of the viral genome. Moreover, none of the patients was treated with antiretroviral drugs such as azidothymidine, tenofovir, and raltegravir, which are known to inhibit XMRV replication and hinder detection of the virus [25,26]. In line with our findings, recent studies showed no evidence of XMRV in HIV-infected patients [27–30]. Thus, the major conclusion of our study is that either HIV infection (and inherent circumstances that exposed individuals to this and other viruses) does not increase the risk of acquiring XMRV infection, or that XMRV does not replicate or persist in peripheral blood cells of HIV patients and does not influence CD4+ T-lymphocyte counts in HIV-positive patients.
Given the long list of studies reporting no occurrence of the XMRV genome in peripheral blood cells (summarized in Table 1), it is unlikely that the virus may persist and replicate in this body compartment. However, the possibility cannot be excluded that XMRV infection causes sporadic local outbreaks and its presence could therefore be restricted to patients with an HIV-unrelated and still unidentified risk factor. Further investigations on different populations and larger number of samples, by using improved PCR assays targeted to different regions of XMRV genome and directed to detect viral and proviral forms of the virus, are needed to determine the real prevalence and distribution of this novel agent.
We declare that we have no conflicts of interest related to this manuscript.