Limits...
No evidence for XMRV in German CFS and MS patients with fatigue despite the ability of the virus to infect human blood cells in vitro.

Hohn O, Strohschein K, Brandt AU, Seeher S, Klein S, Kurth R, Paul F, Meisel C, Scheibenbogen C, Bannert N - PLoS ONE (2010)

Bottom Line: Blood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors.PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene.These results argue against an association between XMRV infection and CFS and MS in Germany.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biological Security 4, Robert Koch-Institute, Berlin, Germany.

ABSTRACT

Background: Xenotropic murine leukemia virus-related virus (XMRV), a novel human retrovirus originally identified in prostate cancer tissues, has recently been associated with chronic fatigue syndrome (CFS), a disabling disease of unknown etiology affecting millions of people worldwide. However, several subsequent studies failed to detect the virus in patients suffering from these illnesses or in healthy subjects. Here we report the results of efforts to detect antibody responses and viral sequences in samples from a cohort of German CFS and relapsing remitting multiple sclerosis (MS) patients with fatigue symptoms.

Methodology: Blood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors. Fatigue severity in MS patients was assessed using the Fatigue Severity Scale (FSS). Validated Gag- and Env-ELISA assays were used to screen sera for XMRV antibodies. PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene. In addition, PBMC cultures were exposed to 22Rv1-derived XMRV to assess infectivity and virus production.

Conclusion: None of the screened sera from CFS and MS patients or healthy blood donors tested positive for XMRV specific antibodies and all PBMC (and PBMC plus LNCaP) cultures remained negative for XMRV sequences by nested PCR. These results argue against an association between XMRV infection and CFS and MS in Germany. However, we could confirm that PBMC cultures from healthy donors and from CFS patients can be experimentally infected by XMRV, resulting in the release of low levels of transmittable virus.

Show MeSH

Related in: MedlinePlus

Lack of infection of XMRV susceptible LNCaP cells by co-culture with activated PBMCs.PCR results with isolated LNCaP cell DNA after co-culture with PBMCs from CFS patients (lane 1–5) and healthy donors (lanes 6–10). Five representative samples out of 10 co-cultures for each group are shown. As control, LNCaP cells were infected with XMRV-containing supernatant from 22Rv1 cells (lane 12). A water-only control (no template control, NTC) was run in lane 11. Results of the GAPDH PCR with the same samples are shown in the lower panel. M  = 100 bp marker.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3008728&req=5

pone-0015632-g003: Lack of infection of XMRV susceptible LNCaP cells by co-culture with activated PBMCs.PCR results with isolated LNCaP cell DNA after co-culture with PBMCs from CFS patients (lane 1–5) and healthy donors (lanes 6–10). Five representative samples out of 10 co-cultures for each group are shown. As control, LNCaP cells were infected with XMRV-containing supernatant from 22Rv1 cells (lane 12). A water-only control (no template control, NTC) was run in lane 11. Results of the GAPDH PCR with the same samples are shown in the lower panel. M  = 100 bp marker.

Mentions: Patient cells were cocultivated with the highly susceptible prostate cancer cell line LNCaP [19] in an attempt to expand, as reported by Lombardi et al [7], possible low levels of infection. Activated PBMC from 10 randomly chosen CFS patients and from 10 healthy donors were added at day three to LNCaP cells for three hours to allow virus transmission. Supernatant from chronically infected 22Rv1 cells was added to separate LNCaP cell cultures as a positive control. After seven days, DNA was isolated from the LNCaP cells and tested by nested XMRV-Gag PCR [5]. The PBMC from neither the 10 CFS patients nor the 10 healthy donors transmitted XMRV to the LNCaP cells (Table 1), whereas incubation with 22Rv1 supernatant resulted in the expected infection of the indicator cells (Fig. 3). Contamination of this DNA preparation with murine genomic DNA or with the pcDNA3.1-VP62 plasmid was ruled out using a PCR designed to detect murine DNA (kindly provided by B. Klempa) and a nested-PCR able to detect less than 10 copies of the pcDNA3.1-VP62 plasmid in 200ng of human DNA (data not shown). This control was necessary because the plasmid is used frequently in many laboratories including our own. Consistent with the PCR results, RT activity was only detected in the supernatants of LNCaP cells incubated with 22Rv1 cell supernatant (Table 1).


No evidence for XMRV in German CFS and MS patients with fatigue despite the ability of the virus to infect human blood cells in vitro.

Hohn O, Strohschein K, Brandt AU, Seeher S, Klein S, Kurth R, Paul F, Meisel C, Scheibenbogen C, Bannert N - PLoS ONE (2010)

Lack of infection of XMRV susceptible LNCaP cells by co-culture with activated PBMCs.PCR results with isolated LNCaP cell DNA after co-culture with PBMCs from CFS patients (lane 1–5) and healthy donors (lanes 6–10). Five representative samples out of 10 co-cultures for each group are shown. As control, LNCaP cells were infected with XMRV-containing supernatant from 22Rv1 cells (lane 12). A water-only control (no template control, NTC) was run in lane 11. Results of the GAPDH PCR with the same samples are shown in the lower panel. M  = 100 bp marker.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3008728&req=5

pone-0015632-g003: Lack of infection of XMRV susceptible LNCaP cells by co-culture with activated PBMCs.PCR results with isolated LNCaP cell DNA after co-culture with PBMCs from CFS patients (lane 1–5) and healthy donors (lanes 6–10). Five representative samples out of 10 co-cultures for each group are shown. As control, LNCaP cells were infected with XMRV-containing supernatant from 22Rv1 cells (lane 12). A water-only control (no template control, NTC) was run in lane 11. Results of the GAPDH PCR with the same samples are shown in the lower panel. M  = 100 bp marker.
Mentions: Patient cells were cocultivated with the highly susceptible prostate cancer cell line LNCaP [19] in an attempt to expand, as reported by Lombardi et al [7], possible low levels of infection. Activated PBMC from 10 randomly chosen CFS patients and from 10 healthy donors were added at day three to LNCaP cells for three hours to allow virus transmission. Supernatant from chronically infected 22Rv1 cells was added to separate LNCaP cell cultures as a positive control. After seven days, DNA was isolated from the LNCaP cells and tested by nested XMRV-Gag PCR [5]. The PBMC from neither the 10 CFS patients nor the 10 healthy donors transmitted XMRV to the LNCaP cells (Table 1), whereas incubation with 22Rv1 supernatant resulted in the expected infection of the indicator cells (Fig. 3). Contamination of this DNA preparation with murine genomic DNA or with the pcDNA3.1-VP62 plasmid was ruled out using a PCR designed to detect murine DNA (kindly provided by B. Klempa) and a nested-PCR able to detect less than 10 copies of the pcDNA3.1-VP62 plasmid in 200ng of human DNA (data not shown). This control was necessary because the plasmid is used frequently in many laboratories including our own. Consistent with the PCR results, RT activity was only detected in the supernatants of LNCaP cells incubated with 22Rv1 cell supernatant (Table 1).

Bottom Line: Blood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors.PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene.These results argue against an association between XMRV infection and CFS and MS in Germany.

View Article: PubMed Central - PubMed

Affiliation: Centre for Biological Security 4, Robert Koch-Institute, Berlin, Germany.

ABSTRACT

Background: Xenotropic murine leukemia virus-related virus (XMRV), a novel human retrovirus originally identified in prostate cancer tissues, has recently been associated with chronic fatigue syndrome (CFS), a disabling disease of unknown etiology affecting millions of people worldwide. However, several subsequent studies failed to detect the virus in patients suffering from these illnesses or in healthy subjects. Here we report the results of efforts to detect antibody responses and viral sequences in samples from a cohort of German CFS and relapsing remitting multiple sclerosis (MS) patients with fatigue symptoms.

Methodology: Blood samples were taken from a cohort of 39 patients fulfilling the Fukuda/CDC criteria (CFS), from 112 patients with an established MS diagnosis and from 40 healthy donors. Fatigue severity in MS patients was assessed using the Fatigue Severity Scale (FSS). Validated Gag- and Env-ELISA assays were used to screen sera for XMRV antibodies. PHA-activated PBMC were cultured for seven days in the presence of IL-2 and DNA isolated from these cultures as well as from co-cultures of PBMC and highly permissive LNCaP cells was analyzed by nested PCR for the presence of the XMRV gag gene. In addition, PBMC cultures were exposed to 22Rv1-derived XMRV to assess infectivity and virus production.

Conclusion: None of the screened sera from CFS and MS patients or healthy blood donors tested positive for XMRV specific antibodies and all PBMC (and PBMC plus LNCaP) cultures remained negative for XMRV sequences by nested PCR. These results argue against an association between XMRV infection and CFS and MS in Germany. However, we could confirm that PBMC cultures from healthy donors and from CFS patients can be experimentally infected by XMRV, resulting in the release of low levels of transmittable virus.

Show MeSH
Related in: MedlinePlus