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

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

Reconstitution of the Ancestral Glycoprotein of Human Endogenous Retrovirus K and Modulation of Its Functional Activity by Truncation of the Cytoplasmic Domain▿ †

Endogenous retroviruses present in the human genome provide a rich record of ancient infections. All presently recognized elements, including the youngest and most intact proviruses of the human endogenous retrovirus K(HML-2) [HERV-K(HML-2)] family, have suffered postinsertional mutations during their time of chromosomal residence, and genes encoding the envelope glycoprotein (Env) have not been spared these mutations. In this study, we have, for the first time, reconstituted an authentic Env of a HERV-K(HML-2) provirus by back mutation of putative postinsertional amino acid changes of the protein encoded by HERV-K113. Aided by codon-optimized expression, we demonstrate that the reconstituted Env regained its ability to be incorporated into retroviral particles and to mediate entry. The original ancient HERV-K113 Env was synthesized as a moderately glycosylated gp95 precursor protein cleaved into surface and transmembrane (TM) subunits. Of the nine N-linked oligosaccharides, four are part of the TM subunit, contributing 15 kDa to its apparent molecular mass of 41 kDa. The carbohydrates, as well as the cytoplasmic tail, are critical for efficient intracellular trafficking, processing, stability, and particle incorporation. Whereas deletions of the carboxy-terminal 6 residues completely abrogated cleavage and virion association, more extensive truncations slightly enhanced incorporation but dramatically increased the ability to mediate entry of pseudotyped lentiviruses. Although the first HERV-K(HML-2) elements infected human ancestors about 30 million years ago, our findings indicate that their glycoproteins are in most respects remarkably similar to those of classical contemporary retroviruses and can still mediate efficient entry into mammalian cells

Human PBMC cultures are susceptible to productive infection by XMRV.

<p>(A) Detection of proviral XMRV sequences by nested PCR in DNAs of PBMC from 5 CSF patients (lanes 1–5) and 5 healthy donors (HD, lanes 6–10) infected for a week with DNase I treated (left hand panel) or DNase I treated and heat-inactivated (right hand panel) supernatants from the XMRV producing 22Rv1 cell line. The no template control (NTC) is in lane 11. DNA prepared from 22Rv1 cells was used as a positive control (lane 12, left panel). Results of a single round PCR for GAPDH are depicted underneath. (B) On the day of DNA isolation, supernatants from the infected PBMC cultures were used to test for virus transmission to LNCaP indicator cells. The results of a nested XMRV PCR with DNA prepared from the exposed LNCaP cells a week after incubation with the PBMC supernatants are shown (lanes 1–10). The control set up was the same as described above (lanes 11 and 12). M  = 100 bp marker. The entire experiment has been repeated twice with similar results.</p

Diagnostic PCR of activated PBMC from CFS and MS patients and healthy donors.

<p>Representative results of nested XMRV PCRs with template DNAs from activated PBMCs after 7 days of culture. Samples from 5 CFS patients (lanes 1–5), 5 MS patients (lanes 6–10) and 5 healthy donors (HD, lanes 11–15) are shown. 200 ng of human genomic DNA spiked with 14 pg of 22Rv1 DNA (genome equivalent of approximately two cells) were used as positive control (lane 16). Results of corresponding single round PCRs for GAPDH as control for DNA integrity and absence of PCR inhibitors are shown in the lower panel. M  = 100 bp marker.</p

Lack of infection of XMRV susceptible LNCaP cells by co-culture with activated PBMCs.

<p>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.</p

Serological assays.

<p>ELISAs with recombinant XMRV proteins were used to detect specific humoral responses. The cut-off was calculated as the mean of all sera from healthy controls plus three times the standard deviation. (A) Results of an XMRV Env antibody ELISA with sera from 36 CFS patients and 17 healthy controls (upper panel) and 112 MS patients and 10 healthy controls (lower panel) identified after unblinding. Sera were incubated at a dilution of 1∶200. (B) Titration of positive control goat sera versus recombinant Gag protein captured by the monoclonal anti-MLV Gag antibody R187. (C) Capture ELISA for the detection of XMRV anti-Gag antibodies in CFS (upper panel) and MS (lower panel) patient sera and healthy controls (gray bars in both panels). Human sera were diluted 1∶200 in blocking buffer.</p

Homozygous mutation in TXNRD1 is associated with genetic generalized epilepsy

Increased oxidative stress has been widely implicated in the pathogenesis in various forms of human epilepsy. Here, we report a homozygous mutation in TXNRD1 (thioredoxin reductase 1) in a family with genetic generalized epilepsy. TXNRD1 is an essential selenium-containing enzyme involved in detoxification of reactive oxygen species (ROS) and redox signaling. The TXNRD1 mutation p.Pro190Leu affecting a highly conserved amino acid residue was identified by whole-exome sequencing of blood DNA from the index patient. The detected mutation and its segregation within the family- all siblings of the index patient were homozygous and the parents heterozygous-were confirmed by Sanger sequencing. TXNRD1 activity was determined in subcellular fractions from a skeletal muscle biopsy and skin fibroblasts of the index patient and the expression levels of the mutated protein were assessed by Se-75 labeling and Western blot analysis. As result of the mutation, the activity of TXNRD1 was reduced in the patient's fibroblasts and skeletal muscle (to 34 +/- 3% and 16 +/- 8% of controls, respectively). In fibroblasts, we detected reduced Se-75-labeling of the enzyme (41 +/- 3% of controls). An in-depth in vitro kinetic analysis of the recombinant mutated TXNRD1 indicated 30-40% lowered k(cat)/Se values. Therefore, a reduced activity of the enzyme in the patient's tissue samples is explained by (i) lower enzyme turnover and (ii) reduced abundance of the mutated enzyme as confirmed by Western blotting and Se-75 labeling. The mutant fibroblasts were also found to be less resistant to a hydrogen peroxide challenge. Our data agree with a potential role of insufficient ROS detoxification for disease manifestation in genetic generalized epilepsy

Harmonizing neuropsychological assessment for mild neurocognitive disorders in Europe

Introduction:  Harmonized neuropsychological assessment for neurocognitive disorders, an international priority for valid and reliable diagnostic procedures, has been achieved only in specific countries or research contexts. Methods:  To harmonize the assessment of mild cognitive impairment in Europe, a workshop (Geneva, May 2018) convened stakeholders, methodologists, academic, and non-academic clinicians and experts from European, US, and Australian harmonization initiatives. Results:  With formal presentations and thematic working-groups we defined a standard battery consistent with the U.S. Uniform DataSet, version 3, and homogeneous methodology to obtain consistent normative data across tests and languages. Adaptations consist of including two tests specific to typical Alzheimer's disease and behavioral variant frontotemporal dementia. The methodology for harmonized normative data includes consensus definition of cognitively normal controls, classification of confounding factors (age, sex, and education), and calculation of minimum sample sizes. Discussion:  This expert consensus allows harmonizing the diagnosis of neurocognitive disorders across European countries and possibly beyond