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Inbred laboratory mouse strains carry endogenous retroviruses (ERVs) classed as ecotropic, xenotropic or polytropic mouse leukemia viruses (E-, X- or P-MLVs). Some of these MLV ERVs produce infectious virus and/or contribute to the generation of intersubgroup recombinants. Analyses of selected mouse strains have linked the appearance of MLVs and virus-induced disease to the strain complement of MLV E-ERVs and to host genes that restrict MLVs, particularly Fv1. Here we screened inbred strain DNAs and genome assemblies to describe the distribution patterns of 45 MLV ERVs and Fv1 alleles in 58 classical inbred strains grouped in two ways: by common ancestry to describe ERV inheritance patterns, and by incidence of MLV-associated lymphomagenesis. Each strain carries a unique set of ERVs, and individual ERVs are present in 5-96% of the strains, often showing lineage-specific distributions. Two ERVs are alternatively present as full-length proviruses or solo long terminal repeats. High disease incidence strains carry the permissive Fv1n allele, tested strains have highly expressed E-ERVs and most have the Bxv1 X-ERV; these three features are not present together in any low-moderate disease strain. The P-ERVs previously implicated in P-MLV generation are not preferentially found in high leukemia strains, but the three Fv1 alleles that restrict inbred strain E-MLVs are found only in low-moderate leukemia strains. This dataset helps define the genetic basis of strain differences in spontaneous lymphomagenesis, describes the distribution of MLV ERVs in strains with shared ancestry, and should help annotate sequenced strain genomes for these insertionally polymorphic and functionally important proviruses., Competing Interests: The authors have declared that no competing interests exist.

Protein A chromatography is an effective capture step to separate Fc-containing biopharmaceuticals from cell culture impurities but is generally not effective for virus removal, which tends to vary among different products. Previous findings have pointed to the differences in feedstocks to protein A, composed of the products and other cell culture-related impurities. To separate the effect of the feedstock components on virus removal, and understand why certain monoclonal antibody (mAb) products have low virus log reduction values (LRVs) across protein A chromatography, we investigated the partitioning of three types of viruses on Eshmuno® A columns. Using pure mAbs, we found that low LRVs were correlated with the presence of the particular mAb product itself, causing altered partitioning patterns. Three virus types were tested, and the trend in partitioning was the same for retrovirus-like particles (RVLPs) expressed in the cell substrate, and its model virus xenotropic murine leukemia virus (XMuLV), whereas slightly different for murine minute virus. These results were extended from previous observation described by Bach and Connell-Crowley (2015) studying XMuLV partitioning on MabSelect SuRe columns, providing further evidence using additional types of viruses and resin. Other product-specific cell culture impurities in harvested cell culture fluid played a lesser role in causing low LRVs. In addition, using high throughput screening (HTS) methods and Eshmuno® A resin plates, we identified excipients with ionic and hydrophobic properties that could potentially alleviate the mAb-induced LRV reduction, indicating that both ionic and hydrophobic interactions were involved. More excipients of such nature or combinations, once optimized, can potentially be used as load and/or wash additives to improve virus removal by protein A. We have demonstrated that HTS is a valuable tool for this type of screening, whether to gain deeper understanding of a mechanism, or to provide guidance during the optimization of protein A process with improved virus removal., (© 2018 Wiley Periodicals, Inc.)

Continuous processing for the production of monoclonal antibodies (mAb) gains more and more importance. Several solutions exist for all the necessary production steps, leading to the possibility to build fully continuous processes. Low pH viral inactivation is a part of the standard platform process for mAb production. Consequently, Klutz et al. introduced the coiled flow inverter (CFI) as a tool for continuous low pH viral inactivation. Besides theoretical calculations of viral reduction, no viral clearance study has been presented so far. In addition, the validation of continuous viral clearance is often neglected in the already existing studies for continuous processing. This study shows in detail the development and execution of a virus study for continuous low pH viral inactivation inside a CFI. The concept presented is also valid for adaptation to other continuous viral clearance steps. The development of this concept includes the technical rationale for an experimental setup, a valid spiking procedure, and finally a sampling method. The experimental results shown represent a viral study using xenotropic murine leukemia virus as a model virus. Two different protein A (ProtA) chromatography setups with varying pH levels were tested. In addition, one of these setups was tested against a batch experiment utilizing the same process material. The results show that sufficient low pH viral inactivation (decadic logarithm reduction value >4) was achieved in all experiments. Complete viral inactivation took place within the first 14.5 min for both continuous studies and the batch study, hence showing similar results. This study therefore represents a successful virus study concept and experiment for a continuous viral inactivation step. Moreover, it was shown that the transfer from batch results to the continuous process is possible. This is accomplished by the narrow residence time distribution of the CFI, showing how close the setup approaches the ideal plug flow and with that batch operation., (© 2018 Wiley Periodicals, Inc.)

Appropriate performance of virus validation studies and testing of unprocessed bulk harvests for retrovirus particle count are procedures in the demonstration of an acceptable level of viral safety for cell-derived biotechnology products. Product-specific validation studies on virus reduction with two model viruses [usually murine leukemia virus (MuLV) and a parvovirus] performed in duplicate runs are standard for clinical trial applications. For the retroviral safety margin, a 6 log reduction is normally expected. Retroviral particle counts are measured traditionally by transmission electron microscopy (TEM) and are commonly performed at contract laboratories. These procedures are quite time-consuming and can be associated with significant costs. In particular, the time factor is a hurdle for companies that want to quickly bring their new products to the clinic. In this session, several strategies on how to lower time, cost, and workload in the evaluation of viral safety for early clinical trial applications, while still ensuring sufficient level of viral safety of the product, were presented. In addition, virus reduction strategies for molecules that do not have the standard antibody structure are presented. Also presented in this session is the feasibility of the use of retrovirus-like particle (RVLP) in the prevalidation of virus removal and the use of quantitative polymerase chain reaction (qPCR) as an alternative to infectivity assays in virus validation studies as well as its use as an alternative to quantitative TEM analysis for determining RVLP count in the bulk harvest of a perfusion bioreactor. LAY ABSTRACT: In this session, several strategies on how to lower time, cost, and workload in the evaluation of viral safety for early clinical trial applications of cell-derived biotechnology products, while still ensuring sufficient level of viral safety of the product, were presented. In addition, virus reduction strategies for molecules that do not have the standard antibody structure are presented. Also presented in this session is the feasibility of the use of retrovirus-like particle (RVLP) in the prevalidation of virus removal and the use of quantitative polymerase chain reaction (qPCR) as an alternative to infectivity assays in virus validation studies as well as its use as an alternative to quantitative TEM analysis for determining RVLP count in the bulk harvest of a perfusion bioreactor., (© PDA, Inc. 2018.)

Naturally-occurring lymphomagenesis is induced by mouse leukemia viruses (MLVs) carried as endogenous retroviruses (ERVs). Replicating the ecotropic MLVs recombines with polytropic (P-ERVs) and xenotropic ERVs (X-ERVs) to generate pathogenic viruses with an altered host range. While most recovered nonecotropic recombinants have a polytropic host range, the X-MLVs are also present in the pre-leukemic tissues. We analyzed two such isolates from the AKR mice to identify their ERV progenitors and to look for evidence of recombination. AKR40 resembles the active X-ERV Bxv1 , while AKR6 has a Bxv1 -like backbone with substitutions that alter the long terminal repeat (LTR) enhancer and the envelope ( env) . AKR6 has a modified xenotropic host range, and its Env residue changes all lie outside of the domain that governs the receptor choice. The AKR6 segment spanning the two substitutions, but not the entire AKR6 env -LTR, exists as an ERV, termed Xmv67 , in AKR, but not in the C57BL/6 mice. This suggests that AKR6 is the product of one, not two, recombination events. Xmv67 originated in the Asian mice. These data indicate that the recombinant X-MLVs that can be generated during lymphomagenesis, describe a novel X-ERV subtype found in the AKR genome, but not in the C57BL/6 reference genome, and identify residues in the envelope C-terminus that may influence the host range.

In the current study we describe a novel DNA sensor system that allows the detection of single catalytic DNA integration events mediated by retrovirus encoded integrase (IN) present in viral particles. This is achieved by rolling circle amplification mediated conversion of enzymatic reactions happening within nanometer dimensions to directly detectable micrometer sized DNA products. The system utilizes the unique integration reaction of IN to generate a surface anchored nicked DNA circle that serves as a substrate for rolling circle amplification and allows for specific, quantitative and sensitive detection of purified recombinant IN or virus particles with a detection limit of less than 30 virus particles per μL of sample. Moreover, by modifying the nucleotide sequences of the utilized DNA it was possible to tailor the system to distinguish between the highly pathogenic lentivirus HIV and the gammaretrovirus murine leukemia virus present in a given sample. Infections with HIV remain a major threat to global health with more than 2 million new infections and 1 million deaths each year. The sensitive and specific detection of HIV particles based on IN activity holds promise for the development of a new type of diagnostic tools suitable for early (within hours of infection) detection of HIV, which would be valuable for prevention strategies as well as for efficient treatment.

Murine leukaemia virus has been suggested to contribute to both autoimmune disease and leukaemia in the NZB mouse and in the (NZB × NZW) F1 (abbreviated B/W) mouse. However, with apparently only xenotropic but no ecotropic virus constitutively expressed in these mice, few mechanisms could explain the aetiology of either disease in either mouse strain. Because pseudotyped and/or inducible ecotropic virus may play a role, we surveyed the ability of murine leukaemia virus in NZB, NZW and B/W mice to infect and form a provirus. From the spleen of NZB mice, we isolated circular cDNA of xenotropic and polytropic virus, which indicates ongoing infection by these viruses. From a B/W lymphoma, we isolated and determined the complete sequence of a putative ecotropic NZW virus. From B/W mice, we recovered de novo endogenous retroviral integration sites (tags) from the hyperproliferating cells of the spleen and the peritoneum. The tagged genes seemed to be selected to aid cellular proliferation, as several of them are known cancer genes. The insertions are consistent with the idea that endogenous retrovirus contributes to B-cell hyperproliferation and progression to lymphoma in B/W mice.

The removal of xenotrpic murine leukemia virus (xMuLV) by size-exclusion filter paper composed of 100% naturally derived cellulose was validated. The filter paper was produced using cellulose nanofibers derived from Cladophora sp. algae. The filter paper was characterized using atomic force microscopy, scanning electron microscopy, helium pycnometry, and model tracer (100 nm latex beads and 50 nm gold nanoparticles) retention tests. Following the filtration of xMuLV spiked solutions, LRV ≥5.25 log10 TCID50 was observed, as limited by the virus titre in the feed solution and sensitivity of the tissue infectivity test. The results of the validation study suggest that the nanocellulose filter paper is useful for removal of endogenous rodent retroviruses and retrovirus-like particles during the production of recombinant proteins., (Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Background: Endogenous murine leukemia retroviruses (MLVs) are high copy number proviral elements difficult to comprehensively characterize using standard low throughput sequencing approaches. However, high throughput approaches generate data that is challenging to process, interpret and present., Results: Next generation sequencing (NGS) data was generated for MLVs from two wild caught Mus musculus domesticus (from mainland France and Corsica) and for inbred laboratory mouse strains C3H, LP/J and SJL. Sequence reads were grouped using a novel sequence clustering approach as applied to retroviral sequences. A Markov cluster algorithm was employed, and the sequence reads were queried for matches to specific xenotropic (Xmv), polytropic (Pmv) and modified polytropic (Mpmv) viral reference sequences., Conclusions: Various MLV subtypes were more widespread than expected among the mice, which may be due to the higher coverage of NGS, or to the presence of similar sequence across many different proviral loci. The results did not correlate with variation in the major MLV receptor Xpr1, which can restrict exogenous MLVs, suggesting that endogenous MLV distribution may reflect gene flow more than past resistance to infection.

Infectivity and reverse transcriptase quantitative real-time polymerase chain reaction (qRT-PCR) assays have been optimized and validated for xenotropic murine leukemia virus (X-MuLV) detection. We have evaluated the assays systematically with regard to specificity, linearity, lower limit of detection (LLOD), lower limit of quantification (LLOQ), and precision. Both assays are specific for X-MuLV detection, with a linear detection range of 0.6-5.6 log(10) TCID(50)/mL for the infectivity assay, and 1.4-6.5 log(10) particles/mL for the qRT-PCR assay. The LLOD and LLOQ of the infectivity and the qRT-PCR assays are determined as 0.5 and 1.0 log(10)/mL, and 1.4 and 2.2 log(10)/mL. The inter-assay repeatability of qRT-PCR assay (4.2% coefficient of variation [CV]) is higher than the infectivity assay (7.9% CV). We have shown that both assays are closely correlated (r = 0.85, P < 0.05, n = 22). The particle/infectivity ratio is determined as 66. Both assays were applied to evaluate virus removal using virus clearance samples of chromatographic and filtration processes. Here, we have demonstrated that the qRT-PCR assay is much faster in testing and is approximately 8-fold more sensitive than the infectivity assay. Therefore, the qRT-PCR assay can replace the infectivity assay in many cases, but both assays are complementary in elucidating the mechanism of virus inactivation and removal in virus clearance validation., (Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Contaminations of cell cultures with microbiological organisms are well documented and can be managed in cell culture laboratories applying reliable detection, elimination and prevention strategies. However, the presence of viral contaminations in cell cultures is still a matter of debate and cannot be determined with general detection methods. In the present study we screened 577 human cell lines for the presence of murine leukemia viruses (MLV). Nineteen cell lines were found to be contaminated with MLV, including 22RV1 which is contaminated with the xenotropic murine leukemia virus-related virus variant of MLV. Of these, 17 cell lines were shown to produce active retroviruses determined by product enhanced reverse transcriptase PCR assay for reverse transcriptase activity. The contaminated cell lines derive from various solid tumor types as well as from leukemia and lymphoma types. A contamination of primary human cells from healthy volunteers could not be substantiated. Sequence analyses of 17 MLV PCR products and five complete MLV genomes of different infected cell lines revealed at least three groups of related MLV genotypes. The viruses harvested from the supernatants of infected cell cultures were infectious to uninfected cell cultures. In the course of the study we found that contamination of human genomic DNA preparations with murine DNA can lead to false-positive results. Presumably, xenotransplantations of the human tumor cells into immune-deficient mice to determine the tumorigenicity of the cells are mainly responsible for the MLV contaminations. Furthermore, the use of murine feeder layer cells during the establishment of human cell lines and a cross-contamination with MLV from infected cultures might be sources of infection. A screening of cell cultures for MLV contamination is recommended given a contamination rate of 3.3%.

Virus filtration (VF) is a key step in an overall viral clearance process since it has been demonstrated to effectively clear a wide range of mammalian viruses with a log reduction value (LRV) > 4. The potential to achieve higher LRV from virus retentive filters has historically been examined using bacteriophage surrogates, which commonly demonstrated a potential of > 9 LRV when using high titer spikes (e.g. 10(10) PFU/mL). However, as the filter loading increases, one typically experiences significant decreases in performance and LRV. The 9 LRV value is markedly higher than the current expected range of 4-5 LRV when utilizing mammalian retroviruses on virus removal filters (Miesegaes et al., Dev Biol (Basel) 2010;133:3-101). Recent values have been reported in the literature (Stuckey et al., Biotech Progr 2014;30:79-85) of LRV in excess of 6 for PPV and XMuLV although this result appears to be atypical. LRV for VF with therapeutic proteins could be limited by several factors including process limits (flux decay, load matrix), virus spike level and the analytical methods used for virus detection (i.e. the Limits of Quantitation), as well as the virus spike quality. Research was conducted using the Xenotropic-Murine Leukemia Virus (XMuLV) for its direct relevance to the most commonly cited document, the International Conference of Harmonization (ICH) Q5A (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland, 1999) for viral safety evaluations. A unique aspect of this work is the independent evaluation of the impact of retrovirus quality and virus spike level on VF performance and LRV. The VF studies used XMuLV preparations purified by either ultracentrifugation (Ultra 1) or by chromatographic processes that yielded a more highly purified virus stock (Ultra 2). Two monoclonal antibodies (Mabs) with markedly different filtration characteristics and with similar levels of aggregate (<1.5%) were evaluated with the Ultra 1 and Ultra 2 virus preparations utilizing the Planova 20 N, a small virus removal filter. Impurities in the virus preparation ultimately limited filter loading as measured by determining the volumetric loading condition where 75% flux decay is observed versus initial conditions (V75). This observation occurred with both Mabs with the difference in virus purity more pronounced when very high spike levels were used (>5 vol/vol %). Significant differences were seen for the process performance over a number of lots of the less-pure Ultra 1 virus preparations. Experiments utilizing a developmental lot of the chromatographic purified XMuLV (Ultra 2 Development lot) that had elevated levels of host cell residuals (vs. the final Ultra 2 preparations) suggest that these contaminant residuals can impact virus filter fouling, even if the virus prep is essentially monodisperse. Process studies utilizing an Ultra 2 virus with substantially less host cell residuals and highly monodispersed virus particles demonstrated superior performance and an LRV in excess of 7.7 log10 . A model was constructed demonstrating the linear dependence of filtration flux versus filter loading which can be used to predict the V75 for a range of virus spike levels conditions using this highly purified virus. Fine tuning the virus spike level with this model can ultimately maximize the LRV for the virus filter step, essentially adding the LRV equivalent of another process step (i.e. protein A or CEX chromatography)., (© 2014 American Institute of Chemical Engineers.)

Background: In 2009, a retrospective study reported the detection of xenotropic murine leukemia virus-related virus (XMRV) in clinical isolates derived from individuals with chronic fatigue syndrome or myalgic encephalomyelitis (CFS). While many efforts to confirm this observation failed, one report detected polytropic murine leukemia virus (pMLV), instead of XMRV. In both studies, Polymerase Chain Reaction (PCR)-based methods were employed which could provide the basis for the development of a practical diagnostic tool. To confirm these studies, we hypothesized that the ability to detect these viruses will not only depend upon the technical details of the methods employed but also on the criteria used to diagnose CFS and the availability of well characterized clinical isolates., Methods: A repository of clinical isolates from geographically distinct sites was generated by the collection of fresh blood samples from well characterized CFS and healthy subjects. Molecular techniques were used to generate assay positive controls and to determine the lower limit of detection (LLOD) for murine retroviral and Intracisternal A particle (Cell 12(4):963-72, 1977) detection methods., Results: We report the establishment of a repository of well-defined, clinical isolates from five, geographically distinct regions of the US, the comparative determination of the LLODs and validation efforts for the previously reported detection methods and the results of an effort to confirm the association of these retroviral signatures in isolates from individuals with CFS in a blinded, multi-site, prospective study. We detected various, murine retroviral DNA signatures but were unable to resolve a difference in the incidence of their detection between isolates from CFS (5/72; 6.7%) and healthy (2/37; 5.4%) subjects (Fisher's Exact Test, p-value = 1). The observed sequences appeared to reflect the detection of endogenous murine retroviral DNA, which was not identical to either XMRV or pMLV., Conclusions: We were unable to confirm a previously reported association between the detection of XMRV or pMLV sequences and CFS in a prospective, multi-site study. Murine retroviral sequences were detected at a low frequency that did not differ between CFS and control subjects. The nature of these sequences appeared to reflect the detection of pre-existing, endogenous, murine retroviral DNA in the PCR reagents employed.

The ability of an anion exchange membrane to purify a γ-retrovirus was assessed and optimised with respect to different loading and wash buffers. Recoveries of infectious virus greater than 50% were consistently obtained, while specific titre was increased up to one thousand fold when compared to the material loaded. Specific proteins removed and retained by this optimised process were identified by mass spectrometry. It was possible to successfully bind and elute the equivalent of 1.27 × 10(8) Ifu/ml of ion exchange membrane. This could then be highly concentrated, with infectious virus concentrated to a maximum of 420-fold compared to the load., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The etiology of fibromyalgia and chronic fatigue syndrome (FM/CFS) is currently unknown. A recurrent viral infection is an attractive hypothesis repeatedly found in the literature since it would explain the persistent pain and tiredness these patients suffer from. The initial striking link of two distinct orphan retroviruses: the gamma retroviruses murine leukemia virus (MLV)-related virus and the delta retrovirus T-lymphotropic virus type 2 (HTLV-2) to chronic fatigue have not been confirmed to date., Results: Genomic DNA (gDNA) from 75 fibromyalgia patients suffering from chronic fatigue and 79 age-matched local healthy controls were screened for the presence of MLV-related and HTLV-2 related proviral sequences. The XMRV env gene was amplified in 20% of samples tested (24% patients/15% healthy controls). Unexpectedly, no PCR amplifications from independent gDNA preparations of the same individuals were obtained. None of the positive samples showed presence of contaminating murine sequences previously reported by other investigators, neither contained additional regions of the virus making us conclude that the initial env amplification came from spurious air-driven amplicon contaminants. No specific HTLV-2 sequences were obtained at any time from any of the 154 quality-controlled gDNA preparations screened., Conclusions: Previous associations between MLV-related or HTLV-2 retrovirus infection with chronic fatigue must be discarded. Thus, studies showing positive amplification of HTLV-2 sequences from chronic fatigue participants should be revised for possible undetected technical problems.To avoid false positives of viral infection, not only extreme precautions should be taken when nested-PCR reactions are prepared and exhaustive foreign DNA contamination controls performed, but also consistent amplification of diverse regions of the virus in independent preparations from the same individual must be demanded.The fact that our cohort of patients did not present evidence of any of the two types of retroviral infection formerly associated to chronic fatigue does not rule out the possibility that other viruses are involved in inciting or maintaining fibromyalgia and/or chronic fatigue conditions.

Retroviral vectors derived from the murine leukemia virus (MuLV) are widely used as the starting material in the development of vectors for gene therapy and critical in answering questions relating to viral pathogenesis. The p30 capsid (CA) is the major viral core protein and an internal group antigen in MuLV. In this study, an enzyme-linked immunosorbent assay (ELISA) was developed for quantitation of MuLV infectious particles with p30 CA core antigen protein. The ELISA was developed using several goat-polyclonal serum against MuLV p30 generated by the NCI as primary antibody and a rat-monoclonal antibody to CA available from ATCC. The MuLV p30 CA antigen was standardized against recombinant MuLV p30 CA expressed from bacteria. The assay is sensitive, accurate and linear within a defined concentration range of CA. Comparison with different MuLV quantitative methods including reporter gene transfer, reverse transcriptase activity assay, and viral RNA quantitative PCR, showed this ELISA protocol to be highly quantifiable within defined ranges, which can be correlated with infectious viral titer., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Polyethylenimine (PEI) was used recently with murine leukemia virus-like particles (MLV-VLPs) to produce a hybrid vector that possesses advantages over the native virus; the transduction efficiency of this vector, however, was less than the transduction efficiency of the native virus. The cause of the reduced efficiency was hypothesized to be related to the involvement of proteins in PEI/MLV-VLP complex formation and overall complex size. To test the hypothesis and potentially improve the efficiency of the hybrid vector, ultracentrifugation and size exclusion chromatography were used to purify MLV-VLP and to study the effect of proteins in cell culture medium on complex formation. Based on dynamic light scattering and electron microscopy, complexes formed from the purified MLV-VLPs were smaller, but surprisingly, less efficient than complexes formed from unpurified MLV-VLPs. The addition of protein to purified MLV-VLPs showed that the initial efficiency could be restored and that the purification strategy was not inactivating the MLV-VLPs. Further, by optimizing the amount of protein added to the purified MLV-VLPs, the level of transduction by PEI/MLV-VLP improved 1.6-fold. Particle characterization showed a correlation between the size of the PEI/MLV-VLP complex and the transduction efficiency, which is likely a result of greater sedimentation and cell contact during in vitro studies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The xenotropic and polytropic mouse leukemia viruses (X-MLVs and P-MLVs, respectively) have different host ranges but use the same functionally polymorphic receptor, XPR1, for entry. Endogenous retroviruses (ERVs) of these 2 gammaretrovirus subtypes are largely segregated in different house mouse subspecies, but both MLV types are found in the classical strains of laboratory mice, which are genetic mosaics of 3 wild mouse subspecies. To describe the subspecies origins of laboratory mouse XP-MLV ERVs and their coevolutionary trajectory with their XPR1 receptor, we screened the house mouse subspecies for known and novel Xpr1 variants and for the individual full-length XP-MLV ERVs found in the sequenced C57BL mouse genome. The 12 X-MLV ERVs predate the origins of laboratory mice; they were all traced to Japanese wild mice and are embedded in the 5% of the laboratory mouse genome derived from the Asian Mus musculus musculus and, in one case, in the <1% derived from M. m. castaneus. While all 31 P-MLV ERVs map to the 95% of the laboratory mouse genome derived from P-MLV-infected M. m. domesticus, no C57BL P-MLV ERVs were found in wild M. m. domesticus. All M. m. domesticus mice carry the fully permissive XPR1 receptor allele, but all of the various restrictive XPR1 receptors, including the X-MLV-restricting laboratory mouse Xpr1(n) and a novel M. m. castaneus allele, originated in X-MLV-infected Asian mice. Thus, P-MLV ERVs show more insertional polymorphism than X-MLVs, and these differences in ERV acquisition and fixation are linked to subspecies-specific and functionally distinct XPR1 receptor variants.

Although novel retroviral vectors for use in gene-therapy products are reducing the potential for formation of replication-competent retrovirus (RCR), it remains crucial to screen products for RCR for both research and clinical purposes. For clinical-grade gammaretrovirus-based vectors, RCR screening is achieved by an extended S(+)L(-) or marker-rescue assay, whereas standard methods for replication-competent lentivirus detection are still in development. In this report, we describe a rapid and sensitive method for replication-competent gammaretrovirus detection. We used this assay to detect three members of the gammaretrovirus family and compared the sensitivity of our assay with well-established methods for retrovirus detection, including the extended S(+)L(-) assay. Results presented here demonstrate that this assay should be useful for gene-therapy product testing.

Distal symmetrical polyneuropathy is the most common form of HIV infection-associated peripheral neuropathy and is often associated with pain. C57BL/6 (B6) mice infected with LP-BM5, a murine retroviral isolate, develop a severe immunodeficiency syndrome similar to that in humans infected with HIV-1, hence the term murine AIDS. We investigated the induction of peripheral neuropathy after LP-BM5 infection in B6 mice. Infected B6 mice, like HIV-infected humans, exhibited behavioral (increased sensitivity to mechanical and heat stimuli) and pathological (transient loss of intraepidermal nerve fibers) signs of peripheral neuropathy. The levels of viral gag RNA were significantly increased in all tissues tested, including spleen, paw skin, lumbar dorsal root ganglia, and lumbar spinal cord, postinfection (p.i.). Correlated with the development of peripheral neuropathy, the tissue levels of several cytokines, including IFN-γ, IL-1β, IL-6, and IL-12, were significantly elevated p.i. These increases had cytokine-specific and tissue-specific profiles and kinetics. Further, treatment with the antiretroviral agent zidovudine either significantly reduced or completely reversed the aforementioned behavioral, pathologic, and cytokine changes p.i. These data suggest that LP-BM5 infection is a potential mouse model of HIV-associated distal symmetrical polyneuropathy that can be used for investigating the roles of various cytokines in infection-induced neuropathic pain. Further investigation of this model could give a better understanding of, and lead to more effective treatments for, HIV infection-associated painful peripheral neuropathy.

The disabling disorder known as chronic fatigue syndrome or myalgic encephalomyelitis (CFS/ME) has been linked in two independent studies to infection with xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (pMLV). Although the associations were not confirmed in subsequent studies by other investigators, patients continue to question the consensus of the scientific community in rejecting the validity of the association. Here we report blinded analysis of peripheral blood from a rigorously characterized, geographically diverse population of 147 patients with CFS/ME and 146 healthy subjects by the investigators describing the original association. This analysis reveals no evidence of either XMRV or pMLV infection. IMPORTANCE Chronic fatigue syndrome/myalgic encephalomyelitis has an estimated prevalence of 42/10,000 in the United States, with annual direct medical costs of $7 billion. Here, the original investigators who found XMRV and pMLV (polytropic murine leukemia virus) in blood of subjects with this disorder report that this association is not confirmed in a blinded analysis of samples from rigorously characterized subjects. The increasing frequency with which molecular methods are used for pathogen discovery poses new challenges to public health and support of science. It is imperative that strategies be developed to rapidly and coherently address discoveries so that they can be carried forward for translation to clinical medicine or abandoned to focus resource investment more productively. Our study provides a paradigm for pathogen dediscovery that may be helpful to others working in this field.

Here I comment on the articles by Lee and colleagues (Retrovirology 2011, 8:82) and Lee and Cho (Retrovirology 2012, 9:23) dealing with an endogenous ecotropic mouse leukemia virus found in C57BL mice.

Many cell lines commonly used for biological studies have been found to harbor exogenous agents such as the human tumor viruses Epstein-Barr virus (EBV) and human papillomavirus. Nevertheless, broad-based, unbiased approaches to globally assess the presence of ectopic organisms within cell model systems have not previously been available. We reasoned that high-throughput sequencing should provide unparalleled insights into the microbiomes of tissue culture cell systems. Here we have used our RNA-seq analysis pipeline, PARSES (Pipeline for Analysis of RNA-Seq Exogenous Sequences), to investigate the presence of ectopic organisms within two EBV-positive B-cell lines commonly used by EBV researchers. Sequencing data sets from both the Akata and JY B-cell lines were found to contain reads for EBV, and the JY data set was found to also contain reads from the murine leukemia virus (MuLV). Further investigation revealed that MuLV transcription in JY cells is highly active. We also identified a number of MuLV alternative splicing events, and we uncovered evidence of APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G)-dependent DNA editing. Finally, reverse transcription-PCR analysis showed the presence of MuLV in three other human B-cell lines (DG75, Ramos, and P3HR1 Cl.13) commonly used by investigators in the Epstein-Barr virus field. We believe that a thorough examination of tissue culture microbiomes using RNA-seq/PARSES-like approaches is critical for the appropriate utilization of these systems in biological studies.

Background: Xenotropic murine leukemia virus (MLV)-related virus (XMRV) and other related MLVs have been described with chronic fatigue syndrome and certain types of prostate cancer. In addition, prevalence rates as high as 7% have been reported in blood donors, raising the risk of transfusion-related transmission. Several laboratories have utilized microneutralization assays as a surrogate marker for detection of anti-MLV serologic responses--with up to 25% of prostate cancer patients reported to harbor neutralizing antibody responses., Study Design and Methods: We developed a high-throughput microneutralization assay for research studies on blood donors using retroviral vectors pseudotyped with XMRV-specific envelopes. Infection with these pseudotypes was neutralized by sera from both macaques and mice challenged with XMRV, but not preimmune serum. A total of 354 plasma samples from blood donors in the Reno/Tahoe area were screened for neutralization., Results: A total of 6.5% of donor samples gave moderate neutralization of XMRV, but not control pseudotypes. However, further testing by Western blot revealed no evidence of antibodies against MLVs in any of these samples. Furthermore, no evidence of infectious virus or viral nucleic acid was observed., Conclusion: A microneutralization assay was developed for detection of XMRV and can be applied in a high-throughput format for large-scale studies. Although a proportion of blood donors demonstrated the ability to block XMRV envelope-mediated infection, we found no evidence that this inhibition was mediated by specific antibodies elicited by exposure to XMRV or MLV. It is likely that this moderate neutralization is mediated through another, nonspecific mechanism., (© 2012 American Association of Blood Banks.)

Background: Xenotropic murine leukemia virus-related virus (XMRV) and polytropic murine leukemia virus (MLV)-related virus are recently described human gammaretroviruses that have been associated with prostate cancer and chronic fatigue syndrome. These studies have been controversial because a number of laboratories have been unable to find evidence of XMRV in similar groups of patients or controls. Because the existence of XMRV raises many questions, we decided to study its presence in a group of patients infected with HIV-1 with a high proportion of intravenous drug use and coinfection by hepatitis C virus., Methods: Forty HIV-1-infected patients under follow-up in our institution were screened for XMRV/MLV by nested polymerase chain reaction using primers targeting the gag and env region. Specific primers for mouse mitochondrial DNA were used to rule out contamination., Results: No evidence of XMRV or polytropic MLV-related sequences was found in any sample from patients or controls. Four samples yielded polymerase chain reaction bands whose sequence corresponded to murine endogenous retroviral sequences, however, contamination with mouse cell DNA was subsequently confirmed., Conclusions: XMRV/MLV viruses do not seem to be associated with HIV-1 infection or intravenous drug use. Contamination of samples or reagents by genomic murine DNA or XMRV vectors could account for the sporadic detection of positive samples for XMRV and related agents.

An understanding in the life cycle of γ-retroviruses has led to significant progress in the development of murine leukemia virus (MLV)-based vectors for gene delivery and human gene therapy. An MLV-based vector consists of the cis-acting sequences important for viral replication and gene expression. The sequence that encodes viral proteins is replaced with the gene of interest. To generate infectious retroviral vectors, viral-encoded proteins are supplied in trans for virion assembly. Here, we describe a method to rapidly generate MLV vectors from transiently transfected human 293T cells. The strategies to purify and titer the vector and to detect the presence of replication competent retrovirus (RCR) in the vector harvest are also described., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Gammaretroviruses related to murine leukemia virus (MLV) have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs) or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.

One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non-infectious retrovirus-like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO ₃⁻, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO ₃⁻ with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO⁻ and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV., (Copyright © 2011 Wiley Periodicals, Inc.)

Genome sequences for Schistosoma japonicum and Schistosoma mansoni are now available. The schistosome genome encodes ~13,000 protein encoding genes for which the function of only a minority is understood. There is a valuable role for transgenesis in functional genomic investigations of these new schistosome gene sequences. In gain-of-function approaches, transgenesis can lead to integration of transgenes into the schistosome genome which can facilitate insertional mutagenesis screens. By contrast, transgene driven, vector-based RNA interference (RNAi) offers powerful loss-of-function manipulations. Our laboratory has focused on development of tools to facilitate schistosome transgenesis. We have investigated the utility of retroviruses and transposons to transduce schistosomes. Vesicular stomatitis virus glycoprotein (VSVG) pseudotyped murine leukemia virus (MLV) can transduce developmental stages of S. mansoni including eggs. We have also observed that the piggyBac transposon is transpositionally active in schistosomes. Approaches with both VSVG-MLV and piggyBac have resulted in somatic transgenesis and have lead to integration of active reporter transgenes into schistosome chromosomes. These findings provided the first reports of integration of reporter transgenes into schistosome chromosomes. Experience with these systems is reviewed herewith, along with findings with transgene mediated RNAi and germ line transgenesis, in addition to pioneering and earlier reports of gene manipulation for schistosomes.

Background: Endogenous retroviruses (ERVs), including murine leukemia virus (MuLV) type-ERVs (MuLV-ERVs), are presumed to occupy ~10% of the mouse genome. In this study, following the identification of a full-length MuLV-ERV by in silico survey of the C57BL/6J mouse genome, its distribution in different mouse strains and expression characteristics were investigated., Results: Application of a set of ERV mining protocols identified a MuLV-ERV locus with full coding potential on chromosome 8 (named ERVmch8). It appears that ERVmch8 shares the same genomic locus with a replication-incompetent MuLV-ERV, called Emv2; however, it was not confirmed due to a lack of relevant annotation and Emv2 sequence information. The ERVmch8 sequence was more prevalent in laboratory strains compared to wild-derived strains. Among 16 different tissues of ~12 week-old female C57BL/6J mice, brain homogenate was the only tissue with evident expression of ERVmch8. Further ERVmch8 expression analysis in six different brain compartments and four peripheral neuronal tissues of C57BL/6J mice revealed no significant expression except for the cerebellum in which the ERVmch8 locus' low methylation status was unique compared to the other brain compartments. The ERVmch8 locus was found to be surrounded by genes associated with neuronal development and/or inflammation. Interestingly, cerebellum-specific ERVmch8 expression was age-dependent with almost no expression at 2 weeks and a plateau at 6 weeks., Conclusions: The ecotropic ERVmch8 locus on the C57BL/6J mouse genome was relatively undermethylated in the cerebellum, and its expression was cerebellum-specific and age-dependent.

Purpose: To investigate the frequency of xenotropic murine leukemia virus (MLV) presence in human cell lines established from mouse xenografts and to search for the evidence of horizontal viral spread to other cell lines., Results: Six of 23 (26%) mouse DNA free xenograft cultures were strongly positive for MLV and their sequences had greater than 99% homology to known MLV strains. Four of five available supernatant fluids from these viral positive cultures were strongly positive for RT activity. Three of these supernatant fluids were studied to confirm the infectivity of the released virions for other human culture cells. Of the 78 non-xenograft derived cell lines maintained in the xenograft culture-containing facilities, 13 (17%) were positive for MLV, including XMRV, a virus strain first identified in human tissues. By contrast, all 50 cultures maintained in a xenograft culture-free facility were negative for viral sequences., Methodology: We examined xenograft tumor cell lines from seven independent laboratories and 128 non-xenografted tumor cell lines. Cell line DNA was examined for mouse DNA contamination, and by 3 Taqman qPCR assays targeting the gag, env or pol regions of MLV. Sequencing was used for viral strain identification. Supernatant fluids were tested for reverse transcriptase (RT) activity., Conclusions: Human cultures derived after mouse xenografting frequently contain and release highly infectious xenotropic MLV viruses. Laboratories working with xenograft-derived human cultures should be aware of the risk of contamination with potentially biohazardous human-tropic mouse viruses and their horizontal spread to other cultures.

Xenotropic murine leukemia virus (MLV)-related virus (XMRV) has been amplified from human prostate cancer and chronic fatigue syndrome (CFS) patient samples. Other studies failed to replicate these findings and suggested PCR contamination with a prostate cancer cell line, 22Rv1, as a likely source. MLV-like sequences have also been detected in CFS patients in longitudinal samples 15 years apart. Here, we tested whether sequence data from these samples are consistent with viral evolution. Our phylogenetic analyses strongly reject a model of within-patient evolution and demonstrate that the sequences from the first and second time points represent distinct endogenous murine retroviruses, suggesting contamination.

Members of the gammaretroviruses--such as murine leukemia viruses (MLVs), most notably XMRV [xenotropic murine leukemia virus (X-MLV)-related virus--have been reported to be present in the blood of patients with chronic fatigue syndrome (CFS). We evaluated blood samples from 61 patients with CFS from a single clinical practice, 43 of whom had previously been identified as XMRV-positive. Our analysis included polymerase chain reaction and reverse transcription polymerase chain reaction procedures for detection of viral nucleic acids and assays for detection of infectious virus and virus-specific antibodies. We found no evidence of XMRV or other MLVs in these blood samples. In addition, we found that these gammaretroviruses were strongly (X-MLV) or partially (XMRV) susceptible to inactivation by sera from CFS patients and healthy controls, which suggested that establishment of a successful MLV infection in humans would be unlikely. Consistent with previous reports, we detected MLV sequences in commercial laboratory reagents. Our results indicate that previous evidence linking XMRV and MLVs to CFS is likely attributable to laboratory contamination.

Background: Autistic spectrum disorder (ASD) is characterized by impaired language, communication and social skills, as well as by repetitive and stereotypic patterns of behavior. Many autistic subjects display a dysregulation of the immune system which is compatible with an unresolved viral infection with prenatal onset, potentially due to vertical viral transmission. Recently, the xenotropic murine leukemia virus-related virus (XMRV) has been implicated in chronic fatigue syndrome (CFS) and in prostate cancer by several, though not all studies., Methodology/principal Findings: We assessed whether XMRV or other murine leukemia virus (MLV)-related viruses are involved in autistic disorder. Using nested PCR targeted to gag genomic sequences, we screened DNA samples from: (i) peripheral blood of 102 ASD patients and 97 controls, (ii) post-mortem brain samples of 20 ASD patients and 17 sex- and age-matched controls, (iii) semen samples of 11 fathers of ASD children, 25 infertile individuals and 7 fertile controls. No XMRV gag DNA sequences were detected, whereas peripheral blood samples of 3/97 (3.1%) controls were positive for MLV. CONCLUSIONS|, Significance: No MLV-related virus was detected in blood, brain, and semen samples of ASD patients or fathers. Hence infection with XMRV or other MLV-related viruses is unlikely to contribute to autism pathogenesis.

Background: Preliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4-6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern., Methodology/principal Findings: To gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus., Conclusions/significance: Our results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied.

Background: The association of xenotropic murine leukemia virus (MLV)-related virus (XMRV) in prostate cancer and chronic fatigue syndrome reported in previous studies remains controversial as these results have been questioned by recent data. Nonetheless, concerns have been raised regarding contamination of human vaccines as a possible source of introduction of XMRV and MLV into human populations. To address this possibility, we tested eight live attenuated human vaccines using generic PCR for XMRV and MLV sequences. Viral metagenomics using deep sequencing was also done to identify the possibility of other adventitious agents., Results: All eight live attenuated vaccines, including Japanese encephalitis virus (JEV) (SA-14-14-2), varicella (Varivax), measles, mumps, and rubella (MMR-II), measles (Attenuvax), rubella (Meruvax-II), rotavirus (Rotateq and Rotarix), and yellow fever virus were negative for XMRV and highly related MLV sequences. However, residual hamster DNA, but not RNA, containing novel endogenous gammaretrovirus sequences was detected in the JEV vaccine using PCR. Metagenomics analysis did not detect any adventitious viral sequences of public health concern. Intracisternal A particle sequences closest to those present in Syrian hamsters and not mice were also detected in the JEV SA-14-14-2 vaccine. Combined, these results are consistent with the production of the JEV vaccine in Syrian hamster cells., Conclusions: We found no evidence of XMRV and MLV in eight live attenuated human vaccines further supporting the safety of these vaccines. Our findings suggest that vaccines are an unlikely source of XMRV and MLV exposure in humans and are consistent with the mounting evidence on the absence of these viruses in humans.

The gammaretroviruses xenotropic murine leukemia virus (MLV)-related virus (XMRV) and MLV have been reported to be more prevalent in plasma and peripheral blood mononuclear cells of chronic fatigue syndrome (CFS) patients than in healthy controls. Here, we report the complex analysis of whole blood and plasma samples from 58 CFS patients and 57 controls from Canada for the presence of XMRV/MLV nucleic acids, infectious virus, and XMRV/MLV-specific antibodies. Multiple techniques were employed, including nested and qRT-PCR, cell culture, and immunoblotting. We found no evidence of XMRV or MLV in humans and conclude that CFS is not associated with these gammaretroviruses.

During pilot studies to investigate the presence of viral RNA of xenotropic murine leukemia virus (MLV)-related virus (XMRV) infection in sera from chronic fatigue syndrome (CFS) patients in Japan, a positive band was frequently detected at the expected product size in negative control samples when detecting a partial gag region of XMRV using a one-step RT-PCR kit. We suspected that the kit itself might have been contaminated with small traces of endogenous MLV genome or XMRV and attempted to evaluate the quality of the kit in two independent laboratories. We purchased four one-step RT-PCR kits from Invitrogen, TaKaRa, Promega and QIAGEN in Japan. To amplify the partial gag gene of XMRV or other MLV-related viruses, primer sets (419F and 1154R, and GAG-I-F and GAG-I-R) which have been widely used in XMRV studies were employed. The nucleotide sequences of the amplicons were determined and compared with deposited sequences of a polytropic endogenous MLV (PmERV), XMRV and endogenous MLV-related viruses derived from CFS patients. We found that the enzyme mixtures of the one-step RT-PCR kit from Invitrogen were contaminated with RNA derived from PmERV. The nucleotide sequence of a partial gag region of the contaminant amplified by RT-PCR was nearly identical (99.4% identity) to a PmERV on chromosome 7 and highly similar (96.9 to 97.6%) to recently identified MLV-like viruses derived from CFS patients. We also determined the nucleotide sequence of a partial env region of the contaminant and found that it was almost identical (99.6%) to the PmERV. In the investigation of XMRV infection in patients of CFS and prostate cancer, researchers should prudently evaluate the test kits for the presence of endogenous MLV as well as XMRV genomes prior to PCR and RT-PCR tests.

Efforts to assess the prevalence of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and chronic fatigue syndrome have relied heavily on PCR-based testing of clinical samples and have yielded widely divergent findings. This week in Retrovirology, reports from four independent research groups illustrate the extreme care needed to exclude DNA or RNA contamination in PCR analyses of XMRV. In addition, phylogenetic evidence suggesting that previously-published XMRV sequences originated from a commonly-used prostate carcinoma cell line (22Rv1) is presented. These findings raise important questions regarding the provenance of XMRV and its potential connection to human disease.

Background: In 2006, a novel gammaretrovirus, XMRV (xenotropic murine leukemia virus-related virus), was discovered in some prostate tumors. A more recent study indicated that this infectious retrovirus can be detected in 67% of patients suffering from chronic fatigue syndrome (CFS), but only very few healthy controls (4%). However, several groups have published to date that they could not identify XMRV RNA or DNA sequences in other cohorts of CFS patients, while another group detected murine leukemia virus (MLV)-like sequences in 87% of such patients, but only 7% of healthy controls. Since there is a high degree of similarity between XMRV and abundant endogenous MLV proviruses, it is important to distinguish contaminating mouse sequences from true infections., Results: DNA from the peripheral blood of 112 CFS patients and 36 healthy controls was tested for XMRV with two different PCR assays. A TaqMan qPCR assay specific for XMRV pol sequences was able to detect viral DNA from 2 XMRV-infected cells (~ 10-12 pg DNA) in up to 5 μg of human genomic DNA, but yielded negative results in the test of 600 ng genomic DNA from 100,000 peripheral blood cells of all samples tested. However, positive results were obtained with some of these samples, using a less specific nested PCR assay for a different XMRV sequence. DNA sequencing of the PCR products revealed a wide variety of virus-related sequences, some identical to those found in prostate cancer and CFS patients, others more closely related to known endogenous MLVs. However, all samples that tested positive for XMRV and/or MLV DNA were also positive for the highly abundant intracisternal A-type particle (IAP) long terminal repeat and most were positive for murine mitochondrial cytochrome oxidase sequences. No contamination was observed in any of the negative control samples, containing those with no DNA template, which were included in each assay., Conclusions: Mouse cells contain upwards of 100 copies each of endogenous MLV DNA. Even much less than one cell's worth of DNA can yield a detectable product using highly sensitive PCR technology. It is, therefore, vital that contamination by mouse DNA be monitored with adequately sensitive assays in all samples tested.

We investigated the prevalence of xenotropic murine leukemia virus-related virus (XMRV) among 293 participants seen at academic hospitals in Boston, Massachusetts. Participants were recruited from the following 5 groups of patients: chronic fatigue syndrome (n = 32), human immunodeficiency virus infection (n = 43), rheumatoid arthritis (n = 97), hematopoietic stem-cell or solid organ transplant (n = 26), or a general cohort of patients presenting for medical care (n = 95). XMRV DNA was not detected in any participant samples. We found no association between XMRV and patients with chronic fatigue syndrome or chronic immunomodulatory conditions.

A xenotropic murine leukemia virus-related virus (XMRV) has recently been reported in association with prostate cancer and chronic fatigue syndrome, with a prevalence of up to 3.7% in the healthy population. We looked for XMRV in 230 patients with human immunodeficiency virus type 1 or hepatitis C infection. XMRV was undetectable in plasma or peripheral blood mononuclear cells by polymerase chain reaction targeting XMRV gag or env. T cell responses to XMRV Gag were undetectable in peripheral blood mononuclear cells by ex vivo gamma interferon enzyme-linked immunospot assay. In our cohorts, XMRV was not enriched in patients with blood-borne or sexually transmitted infections from the United Kingdom and Western Europe.

Background: There are questions regarding the prevalence of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and its association with the RNASEL R462Q polymorphism. We therefore investigated whether XMRV infection could be found in patients with prostate cancer from the southern United States, and we sought to verify the association with the R462Q., Methods: Prostate tissue specimens of 144 patients with prostate cancer from the southern United States were genotyped for R462Q by real time polymerase chain reaction allelic discrimination and were screened for XMRV proviral DNA by nested polymerase chain reaction specific for the env gene., Results: The R462Q polymorphism was found at an allelic frequency of 0.33. XMRV was detected in 32 (22%) of the 144 patients. Patients were significantly more likely to test positive for XMRV in both tumor and normal tissue rather than either alone (κ = 0.64). A positive result for XMRV was not significantly correlated with the R462Q polymorphism (P = .82) or clinical pathological parameters of prostate cancer, including Gleason score (P = .29)., Conclusions: XMRV is detectable in normal and tumor prostate tissue from patients with prostate cancer, independent of R462Q. The presence of XMRV in normal tissue suggests that infection may precede cancer onset.

A mass spectrometry (MS) approach was used to analyze viral core proteins of the murine leukemia virus (MuLV)-based gene delivery vector. The retroviral particles produced by traditional methods were concentrated and purified by ultracentrifugation and spin column for matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) MS. MALDI application detected all core MuLV proteins, partial degradation of p10, phosphorylation of p12, as well as the previously unknown formation of a polymeric supramolecular complex between p15 and p30 core proteins. ESI provided information on the post-translational modifications of MuLV core proteins. Data suggest myristoylation of p15 and oxidation of methionine residues in both p12 and p30, whereas cysteine residues in p10, p15 and p30 were not oxidized. The current study demonstrates that MALDI and ESI are efficient tools for viral core protein analysis and can be used as analytical tools in virology and biotechnology of gene delivery vectors., (Copyright © 2010. Published by Elsevier B.V.)