Prevalence of Xenotropic Murine Leukemia Virus-Related Virus Infection in Different Risk Populations in Spain

Abstract

Human infection with the xenotropic murine leukemia virus-related virus (XMRV) has been associated controversially with prostate cancer and chronic fatigue syndrome. Information is lacking about the mechanisms of transmission and potential risk groups for XMRV infection. Plasma and peripheral blood mononuclear cells (PBMCs) from individuals with retroviral infections, chronic viral hepatitis, autoimmune diseases, prostate cancer, chronic fatigue syndrome, and blood donors were tested for XMRV markers. Antibodies to XMRV proteins p15E and gp70 were examined using research assays. DNA extracted from PBMCs was tested for the presence of XMRV gag and env sequences. A total of 1103 specimens belonging to individuals with chronic fatigue syndrome and/or fibromyalgia (437), prostate cancer (69), HIV-1 (149), HTLV-1/2 (31), chronic hepatitis B (81), chronic hepatitis C (72), autoimmune diseases (18), and blood donors (246) were examined. Overall, three samples (0.3%) were p15E seroreactive (two HTLV-1 and one HCV patient). Another 15 (1.4%) were gp70 seroreactive (six chronic fatigue syndrome-fibromyalgia, four blood donors, two HIV-1, one prostate cancer, one HBV, and one HCV). Four specimens were initially positive for XMRV gag sequences, but none could be confirmed by repeated testing. In summary, no evidence of XMRV infection was found in populations with retroviral and viral hepatitis infections in Spain. Likewise, XMRV was not recognized in patients with autoimmune diseases, chronic fatigue syndrome-fibromyalgia, prostate cancer, or healthy blood donors.

Introduction

The discovery of xenotropic murine leukemia virus-related virus (XMRV), a new human retrovirus, and its association with prostate cancer (PrCa)¹ and chronic fatigue syndrome (CFS)² have generated enormous interest. Different studies have examined the association of XMRV with human illnesses, with conflicting results. While a link with PrCa has been shown in some studies examining either tumor tissue or blood,^1,3
–5 others have not confirmed these findings.^6
–8 Likewise, the association of XMRV with CFS has been controversial since the first report claiming that two-thirds of patients were infected with XMRV was published in 2009.² Many studies conducted within the past year have not confirmed this link, including reports from Western Europe,^9
–11 the United States,^12,13 and China.¹⁴ More recently, an intriguing result was reported by researchers at the National Institutes of Health who identified gene sequences closely related to polytropic murine leukemia viruses (MLVs) in the blood of patients with CFS.¹⁵

Following the initial enthusiasm, even in the absence of knowledge about the mechanisms of transmission¹⁶ and pathogenicity, several reports related to the in vitro activity of antiretroviral agents against XMRV have appeared.^17
–19 Moreover, trials evaluating the clinical efficacy of these compounds in patients with CFS have been initiated.

Given that XMRV is a human retrovirus and potentially may share routes of transmission similar to other human retroviruses, such as HIV or HTLV, we conducted a cross-sectional study in Spain using serologic and molecular assays to screen for XMRV infection in blood from individuals with distinct retroviral infections and viral hepatitis, as well as illnesses potentially linked to retroviral infections, including autoimmune diseases, CFS, and PrCa.

Materials and Methods

Study population

Stored plasma and peripheral blood mononuclear cells (PBMCs) from individuals chronically infected with HIV-1, HTLV-1 or HTLV-2, hepatitis B virus (HBV), or hepatitis C virus (HCV) were identified at the repository of our institution. Likewise, specimens from patients with autoimmune diseases were obtained from Hospital Germans Trias i Pujol, located in Barcelona. Samples from patients with PrCa were obtained from Hospital La Paz in Madrid. Specimens belonging to patients diagnosed with CFS and/or fibromyalgia were obtained from the National DNA Repository located in Salamanca, although a subset was collected at our institution from subjects seen in 2010. Finally, healthy blood donors were provided by blood banks located in Madrid. All patients with a diagnosis of CFS met the criteria of the 1994 International CFS Research Case Definition.²⁰ The study was approved by the hospital ethics committee and all patients gave written informed consent for genetic testing.

Serologic assays

Plasma samples were evaluated for the presence of antibodies to XMRV transmembrane protein p15E and envelope protein gp70 using prototype ARCHITECT XMRV research assays (Abbott Diagnostics, Abbott Park, IL).²¹ Moreover, investigational Western blot (WB) assays using purified XMRV viral lysate as well as recombinant gp70 protein were used to confirm initially seroreactive samples.²¹ Inhibition studies using recombinant proteins were performed in order to support the presence of proteins migrated in the WB strips.

Nested polymerase chain reaction assay

DNA extracted using a commercial assay (Qiagen) from PBMCs was tested for the presence of XMRV gag sequences using nested polymerase chain reaction (PCR) with primer sets and conditions originally described by Urisman et al.¹ and Lombardi et al.² (first round: 419F/1154R; second round: GAG-I-F/GAG-I-R). PCR was performed using 100–250 ng of total nucleic acid from PBMCs and 25 μl Taq DNA polymerase (Promega; Madison, WI). Samples that gave positive results were further evaluated using a nested PCR for XMRV env sequences with primer sets 4166F/7622 and 5922F/6273, and conditions previously reported elsewhere.²

The XMRV-positive PrCa cell line, 22Rv1, was used as a positive control. To minimize the risk of contamination, all experiments were performed under strict conditions to avoid contamination, including use of distinct reagents in different laboratory rooms. Negative controls were included in each experiment.

The sensitivity of the nested PCR assay for detecting XMRV sequences was tested using 10-fold serial dilutions (1 to 10⁻⁹) of 22Rv1 PBMC DNA in water and negative background DNA considering the known level production of XMRV from the 22Rv1 PrCa cell line.²² The detection limit was 3–5 copies per reaction. Given that the exact integrated copy number of XMRV proviral DNA in 22Rv1 in the prostate cancer cell line is unknown, our sensitivity threshold is only approximate and based on serial dilutions. The use of plasmids of known copy number of XMRV sequences as controls would provide a better estimation of the sensitivity of our technique.

Sequence analysis

XMRV sequences were obtained using the ViroSeq HIV-1 Genotyping System (Abbott Diagnostics, Madrid, Spain). The rate of similarity between sequences was analyzed using BLAST. The phylogenetic analysis was performed using a multiple sequence alignment program with Clustal W2.

Results

Study population

Plasma and PBMCs were collected from a total of 1103 individuals: 237 with CFS, 100 with CFS and fibromyalgia, 100 with fibromyalgia, 69 with PrCa, 149 HIV-1 infected, 24 HTLV-1 infected, 7 HTLV-2 infected, 81 with chronic hepatitis B, 72 with chronic hepatitis C, 18 with autoimmune diseases, and 246 healthy blood donors. Demographics data for the different cohorts are summarized in Table 1.

Table 1.

Characteristics of the Study Population

Category	No.	Median age (years)	Female gender (%)	Country of origin (%)
Chronic fatigue syndrome	237	46 (22–78)	86.1	Spain (100)
Fibromyalgia	100	51 (35–71)	100	Spain (100)
Chronic fatigue syndrome and fibromyalgia	100	47 (18–76)	94	Spain (100)
Prostate cancer	69	64 (51–81)	0	Spain (100)
HIV-1	149	46 (21–69)	16	Spain (82), South America (14), Africa (2), Rest of Europe (2)
HTLV-1	24	48 (10–66)	65	South America (77), Spain (13), Africa (10)
HTLV-2	7	42 (33–52)	0	Spain (86), Africa (14)
Chronic hepatitis B	81	49 (23–63)	8	Spain (92), South America (8)
Chronic hepatitis C	72	45 (22–59)	45	Spain (93), South America (7)
Autoimmune diseases	18	46 (26–61)	82	Spain (100)
Healthy blood donors	246	39 (18–62)	26	Spain (96), South America (3), rest of Europe (1)
Total	1103	44 (20–64)	47	Spain (94)

The majority of individuals with CFS were female (86.1%) and the average age was 46 years. All patients with fibromyalgia were women with a mean age of 51 years. Individuals with both CFS and fibromyalgia were mostly women (94%) with a mean age of 47 years old. The most common symptoms of patients with CFS were recurrent headache (82%), nonrestorative sleep (98%), chronic pain (79%), muscle weakness (93%), and extreme exhaustion lasting more than 24 h after physical exercise (98%).

The mean age of patients with PrCa was 64 years old. The mean prostate serum antigen titer was 8.9 (1.8–65) ng/ml at the time of diagnosis. The tumor clinical stage was T1 in 84.6%, T2 in 9.6%, T3 in 3.8%, and T4 in 1.9%.

Serological reactivity

Table 2 summarizes the results of the XMRV antibody screening. Three (0.3%) of 1103 plasma specimens were seroreactive to p15E. The sample/cut-off (S/CO) was >1.0 in two individuals infected with HTLV-1 (1.2 and 4.8, respectively) and one with chronic hepatitis C (1.04).

Table 2.

ARCHITECT gp70 and p15E Antibody Reactivity

Category	No.	XMRV p15E Ab⁺ No. (%)	XMRV gp70 Ab⁺ No. (%)
Chronic fatigue syndrome	237	0	6 (2.5)
Fibromyalgia	100	0	0
Chronic fatigue syndrome and fibromyalgia	100	0	0
Prostate cancer	69	0	1 (1.4)
HIV-1	149	0	2 (1.3)
HTLV-1	24	2 (8.3)	0
HTLV-2	7	0	0
Chronic hepatitis B	81	0	1 (1.2)
Chronic hepatitis C	72	1 (1.4)	1 (1.4)
Autoimmune diseases	18	0	0
Healthy blood donors	246	0	4 (1.6)
Total	1103	3 (0.3)	15 (1.4)

A total of 15 (1.4%) of 1103 plasma samples were gp70 seroreactive. The S/CO values were >1.0 in 6/237 patients with CFS (1.12, 1.13, 1.69, 1.88, 5.63, and 10.53, respectively), 1/69 patients with PrCa (1.74), 2/149 with HIV-1 infection (1.00 and 49.05, respectively), 1/81 with chronic hepatitis B (5.24), 1/72 with chronic hepatitis C (3.22), and in 4/246 healthy blood donors (1.15, 1.43, 2.16, and 3.35, respectively).

All p15E and gp70 antibody reactive samples were tested using investigational WBs utilizing XMRV viral lysate proteins or recombinant XMRV gp70. Two HTLV-1 patients were p15E positive in the ARCHITECT assay and p15E WB reactive. Nine of the ARCHITECT gp70 positive samples were also WB reactive for gp70. Two subjects (one HIV-1 and one blood donor) were reactive only to gp70. Four subjects (one CFS, one PrCa, and two blood donors) were gag p30 and gp70 reactive. One CFS patient had gag p15, gag p30, and gp70 reactivity. One CFS patient was p15E and gp70 reactive on WB and another CFS patient was gag p15 and gp70 reactive (Table 3).

Table 3.

Demographic, Serologic, and Genetic Profile of Individuals Positive for XMRV Markers

Patient no.	Risk population	Age (years)	Gender	Origin	Risk group	p15E Ab S/CO	gp70 Ab S/CO	WB lysate	Nested PCR (gag)	Nested PCR (env)
1	HCV	38	M	Spain	IDU	1.04	0.05	Neg	Neg	Neg
2	HTLV-1	48	F	Colombia	None	4.80	0.38	p15E, +/− gp70	Neg	Neg
3	HTLV-1	10	M	Ethiopia	None	1.20	0.04	p15E^a	Neg	Neg
4	CFS	40	F	Spain	None	0.18	1.88	Neg	Neg	Neg
5	CFS	48	F	Spain	None	0.17	10.58	p15,^b p30, gp70	Neg	Neg
6	CFS	36	F	Spain	None	0.15	1.12	p15,^c gp70	Neg	Neg
7	CFS	37	F	Spain	None	0.16	5.63	p30, gp70	Neg	Neg
8	CFS	38	F	Spain	None	0.15	1.69	p15E,^d gp70	Neg	Neg
9	CFS	53	M	Spain	None	0.18	1.13	Neg	Neg	Neg
10	PrCa	81	M	Spain	Transfusion	0.25	1.74	p30, gp70	Pos	Neg
11	HIV-1	44	M	Spain	IDU	0.20	1.00	gp70	Neg	Neg
12	HIV-1	48	M	Argentina	MSM	0.23	49.05	Neg	Neg	Neg
13	HCV	55	F	Spain	IDU	0.14	3.22	Neg	Neg	Neg
14	HBV	47	M	Spain	None	0.18	5.24	Neg	Neg	Neg
15	Donor	36	M	Spain	None	0.24	3.35	p30, gp70	Neg	Neg
16	Donor	50	F	Spain	None	0.25	2.16	p30, gp70	Neg	Neg
17	Donor	47	M	Spain	None	0.24	1.43	gp70	Neg	Neg
18	Donor	32	M	Spain	None	0.22	1.15	Neg	Neg	Neg

Inhibition study using recombinant proteins supported the presence of p15E.

Inhibition study using recombinant proteins supported the presence of gag p15.

Inhibition data suggested evidence of gag p15.

Inhibition data suggested evidence of p15E.

M, male; F, female; CFS, chronic fatigue syndrome; PrCa, prostate cancer; HCV, hepatitis C virus; HBV, hepatitis B virus; S/CO, sample/cut-off ratio; IDU, intravenous drug user; MSM, men who have sex with men.

XMRV gene sequences

PBMCs from 872 out of the 1103 subjects were screened for the presence of XMRV DNA using nested PCR targeting gag. Amplification products of the expected size were observed in four out of the 872 reactions. Interestingly, all were patients with PrCa (4/69; 5.8%). The clinical stage of these tumors was T1 in two, T2 in one, and T3 in the remaining subject. Genetic sequencing of the PCR products revealed that only one was homologous [99.7% to VP62 (GenBank accession number, NC_007815.1)] to XMRV sequences reported to date, with the other three being human genome sequences. XMRV env sequences could not be amplified from any of these four patients.

XMRV gag sequences were not amplified from any of the patients with p15E antibody reactivity. Only one patient with PrCa had both gp70 antibody reactivity and detectable XMRV gag sequences that were confirmed by phylogenetic analysis. This patient had advanced PrCa stage (T3) and a prostate serum antigen of 36.6 ng/ml. However, repeated testing of a separate specimen could not reproduce the original results.

Discussion

In the present study, the prevalence of XMRV infection was assessed in samples collected from 1103 individuals in Spain with retroviral infections, chronic viral hepatitis, CFS/fibromyalgia, autoimmune diseases, PrCa, and healthy blood donors. Serologic screening using prototype assays showed a low seroprevalence of antibodies to XMRV: 0.3% p15E reactive and 1.4% gp70 reactive. Notably, none of the subjects had antibodies to both XMRV p15E and gp70. Parallel analysis of PBMCs for the presence of XMRV proviral sequences was consistent with very low rates of detectable XMRV in this study population. In fact, the only subject with initially detectable XMRV gag sequences was a patient with PrCa. However, results could not be confirmed by retesting a separate specimen of this patient.

Based on combined serologic and molecular screening, only 1 out of 69 (1.4%) PrCa patients in this study had evidence of XMRV infection. This is somewhat in agreement with several other European studies examining PrCa^6
–8 but contrasts with other studies that have claimed greater prevalence rates.^1,3
–5 It should be acknowledged that most prior studies associating XMRV with PrCa have screened prostate tissue rather than PBMC, as used in the present study. It is conceivable that XMRV levels may be lower in the PBMC compartment as compared to prostate tissue. Notably, in experimentally infected rhesus macaques, detection of XMRV provirus in PBMCs was transient.²³ Moreover, data from several studies indicate a stronger association between XMRV and familial PrCa than XMRV and sporadic PrCa.^1,4,5 Familial versus sporadic PrCa was not stratified in the present study but the proportion of familial cases was likely quite low.

The PrCa patient that initially had both positive serology and molecular evidence of XMRV infection was diagnosed with PrCa at 81 years of age, presenting with a locally advanced tumor stage (T3). He is white with a prior history of laryngeal cancer but denied intravenous drug use or homosexual relationships. The patient had resided in West Sahara during his younger years. Of interest, this individual was transfused in Spain more than a decade earlier. No other risk factors for XMRV infection could be identified for this individual.

It is noteworthy that XMRV infection was not detected in any of the 437 individuals diagnosed with CFS and/or fibromyalgia. This result is in agreement with reports from several other studies^9

–14 but clearly contrasts with data from Lombardi et al.² showing evidence of XMRV infection in 67% of CFS patients.

With the possible exception of the HTLV-1-infected cohort, there was no evidence of an increased XMRV seroprevalence in any specific risk group. Two of 24 HTLV-1-infected patients were p15E seropositive but harbored no detectable XMRV in PBMCs. Of note, occasional reactivity in the p15E assay has been observed for a larger cohort of HTLV-1-infected patients, apparently due to cross-reactivity (X. Qiu, unpublished observation). The significance of isolated reactivity to gp70 in a few blood donors in our study is currently unknown, but has previously been described by others testing large numbers of blood donors, all of whom were negative for XMRV sequences.²⁴

Several reasons have been postulated to explain the large discrepancy between the results of studies that have examined the prevalence of XMRV infection in distinct human populations. Differences in the geographic distribution of XMRV infections could account for some of the conflicting observations.⁷ Contamination also has the potential to confound results. Possible sources of contamination include the presence of MLV-related nucleic acids in some commercial PCR reagents containing mouse monoclonal antibodies, traces of mouse DNA in human blood and tissue samples, XMRV-positive control reagents, and nucleic acids from XMRV-infected human tumor cell lines.^25

–30 In addition, new data about contamination were recently reported stressing that the source of XMRV could be a recombinant virus generated in the laboratory a few decades ago.^31,32

Technical differences related to the assays used to screen for XMRV could also account for some of the conflicting results.^33

–36 The lack of highly sensitive and well-standardized methods to detect XMRV infection coupled with a lack of widely available positive human control specimens has most certainly contributed to the controversy. The PCR amplification strategy used in the present study utilized identical primer combinations and conditions used previously to successfully detect XMRV.^1,2 The quantity of input DNA could represent a limitation in that higher quantities of template DNA have been shown to increase the efficiency of XMRV detection.⁵ Thus, it is theoretically possible that DNA proviral levels in at least some samples are below the threshold of detection. However, the methods in the present study are comparable to those used previously to successfully detect XMRV.^1,2

In summary, we found no evidence of XMRV infection in a variety of risk groups in Spain, including patients with CFS, retroviral infections (HIV and HTLV), chronic viral hepatitis B and C, autoimmune diseases, and PrCa.

Footnotes

Acknowledgments

We would like to thank Natalia Zahonero, Patricia Parra, Dr. Pablo Labarga, Pilar Torres, Luz Barbolla, and Dr. Ferran Garcia-Fructuoso for excellent technical assistance and/or fruitful discussions. We appreciate the kind provision of specimens by the FSGCDB Group (Fibromyalgia and SFC Spanish Genetic and Clinical Data Bank), and particularly wish to thank the following researchers: Jordi Carbonell (Fibromialgia Unit, Hospital del Mar, Barcelona), Javier Rivera (Fibromialgia Unit, Hospital Gregorio Marañon, Madrid), Jose Alegre (Chronic Fatigue Unit, Hospital Vall d'Hebrón, Barcelona), Javier Vidal (Rheumatology Service, Hospital General, Guadalajara), and Antonio Collado (Fibromialgia Unit, Hospital Clinic, Barcelona).

This work was supported in part by grants from Ministerio Ciencia y Tecnología (SAF2010-22232), Fundación Investigación y Educación en SIDA, Red de Investigación en SIDA (RIS, RD06/0006), and the European NEAT project.

Author Disclosure Statement

J. Hackett, Jr., X. Qui, and P. Swanson are employees and shareholders of Abbott Laboratories.

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